Antigen-binding molecule containing modified antibody variable region

ABSTRACT

The present inventors have successfully prepared an antigen-binding molecule comprising an antibody variable region that has binding activity against a molecule expressed on the surface of a T cell and a molecule expressed on the surface of any other immunocyte, but does not bind to these molecules at the same time. The present invention allows the preparation of an antigen-binding molecule capable of circumventing adverse reactions that may be caused by the cross-linking of T cells to other immunocytes, and provides an antigen-binding molecule suitable as a drug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/035,098, filed on May 6, 2016, which is the National Stage ofInternational Application No. PCT/JP2014/079785, filed on Nov. 11, 2014,which claims the benefit of Japanese Application No. JP 2013-232803,filed on Nov. 11, 2013.

TECHNICAL FIELD

The present invention provides an antigen-binding molecule comprising:an antibody variable region that is capable of binding to two differentantigens (first antigen and second antigen), but does not bind to theseantigens at the same time; and an antibody variable region binding to athird antigen different from these antigens, a pharmaceuticalcomposition comprising the antigen-binding molecule, and a method forproducing the antigen-binding molecule.

BACKGROUND ART

Antibodies have received attention as drugs because of having highstability in plasma and producing few adverse reactions (Nat.Biotechnol. (2005) 23, 1073-1078 (Non Patent Literature 1) and Eur JPharm Biopharm. (2005) 59 (3), 389-396 (Non Patent Literature 2)). Theantibodies not only have an antigen-binding effect and an agonist orantagonist effect, but induce cytotoxic activity mediated by effectorcells (also referred to as effector functions), such as ADCC (antibodydependent cytotoxicity), ADCP (antibody dependent cell phagocytosis), orCDC (complement dependent cytotoxicity). Particularly, antibodies ofIgG1 subclass exhibit the effector functions for cancer cells.Therefore, a large number of antibody drugs have been developed in thefield of oncology.

For exerting the ADCC, ADCP, or CDC of the antibodies, their Fc regionsmust bind to antibody receptors (FcγR) present on effector cells (suchas NK cells or macrophages) and various complement components. Inhumans, FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms havebeen reported as the protein family of FcγR, and their respectiveallotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (NonPatent Literature 3)). Of these isoforms, FcγRIa, FcγRIIa, and FcγRIIIahave, in their intracellular domains, a domain called ITAM(immunoreceptor tyrosine-based activation motif), which transducesactivation signals. By contrast, only FcγRIIb has, in its intracellulardomain, a domain called ITIM (immunoreceptor tyrosine-based inhibitorymotif), which transduces inhibition signals. These isoforms of FcγR areall known to transduce signals through cross-linking by immune complexesor the like (Nat. Rev. Immunol. (2008) 8, 34-47 (Non Patent Literature4)). In fact, when the antibodies exert effector functions againstcancer cells, FcγR molecules on effector cell membranes are clustered bythe Fc regions of a plurality of antibodies bound onto cancer cellmembranes and thereby transduce activation signals through the effectorcells. As a result, a cell-killing effect is exerted. In this respect,the cross-linking of FcγR is restricted to effector cells located nearthe cancer cells, showing that the activation of immunity is localizedto the cancer cells (Ann. Rev. Immunol. (1988). 6. 251-81 (Non PatentLiterature 5)).

Naturally occurring immunoglobulins bind to antigens through theirvariable regions and bind to receptors such as FcγR, FcRn, FcαR, andFcεR or complements through their constant regions. Each molecule ofFcRn (binding molecule that interacts with an IgG Fc region) binds toeach heavy chain of an antibody in a one-to-one connection. Hence, twomolecules of FcRn reportedly bind to one IgG-type antibody molecule.Unlike FcRn, etc., FcγR interacts with an antibody hinge region and CH2domains, and only one molecule of FcγR binds to one IgG-type antibodymolecule (J. Bio. Chem., (20001) 276, 16469-16477). For the bindingbetween FcγR and the Fc region of an antibody, some amino acid residuesin the hinge region and the CH2 domains of the antibody and sugar chainsadded to Asn 297 (EU numbering) of the CH2 domains have been found to beimportant (Chem. Immunol. (1997), 65, 88-110 (Non Patent Literature 6),Eur. J. Immunol. (1993) 23, 1098-1104 (Non Patent Literature 7), andImmunol. (1995) 86, 319-324 (Non Patent Literature 8)). Fc regionvariants having various FcγR-binding properties have previously beenstudied by focusing on this binding site, to yield Fc region variantshaving higher binding activity against activating FcγR (WO2000/042072(Patent Literature 1) and WO2006/019447 (Patent Literature 2)). Forexample, Lazar et al. have successfully increased the binding activityof human IgG1 against human FcγRIIIa (V158) to approximately 370 timesby substituting Ser 239, Ala 330, and Ile 332 (EU numbering) of thehuman IgG1 by Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci.U.S.A. (2006) 103, 4005-4010 (Non Patent Literature 9) and WO2006/019447(Patent Literature 2)). This altered form has approximately 9 times thebinding activity of a wild type in terms of the ratio of FcγRIIIa toFcγIIb (A/I ratio). Alternatively, Shinkawa et al. have successfullyincreased binding activity against FcγRIIIa to approximately 100 timesby deleting fucose of the sugar chains added to Asn 297 (EU numbering)(J. Biol. Chem. (2003) 278, 3466-3473 (Non Patent Literature 10)). Thesemethods can drastically improve the ADCC activity of human IgG1 comparedwith naturally occurring human IgG1.

A naturally occurring IgG-type antibody typically recognizes and bindsto one epitope through its variable region (Fab) and can therefore bindto only one antigen. Meanwhile, many types of proteins are known toparticipate in cancer or inflammation, and these proteins may crosstalkwith each other. For example, some inflammatory cytokines (TNF, IL1 andIL6) are known to participate in immunological disease (Nat. Biotech.,(2011) 28, 502-10 (Non Patent Literature 11)). Also, the activation ofother receptors is known as one mechanism underlying the acquisition ofdrug resistance by cancer (Endocr Relat Cancer (2006) 13, 45-51 (NonPatent Literature 12)). In such a case, the usual antibody, whichrecognizes one epitope, cannot inhibit a plurality of proteins.

Antibodies that bind to two or more types of antigens by one molecule(these antibodies are referred to as bispecific antibodies) have beenstudied as molecules inhibiting a plurality of targets. Binding activityagainst two different antigens (first antigen and second antigen) can beconferred by the modification of naturally occurring IgG-type antibodies(mAbs. (2012) Mar. 1, 4 (2)). Therefore, such an antibody has not onlythe effect of neutralizing these two or more types of antigens by onemolecule but the effect of enhancing antitumor activity through thecross-linking of cells having cytotoxic activity to cancer cells. Amolecule with an antigen-binding site added to the N or C terminus of anantibody (DVD-Ig and scFv-IgG), a molecule having different sequences oftwo Fab regions of an antibody (common L-chain bispecific antibody andhybrid hybridoma), a molecule in which one Fab region recognizes twoantigens (two-in-one IgG), and a molecule having a CH3 domain loop asanother antigen-binding site (Fcab) have previously been reported asmolecular forms of the bispecific antibody (Nat. Rev. (2010), 10,301-316 (Non Patent Literature 13) and Peds (2010), 23 (4), 289-297 (NonPatent Literature 14)). Since any of these bispecific antibodiesinteract at their Fc regions with FcγR, antibody effector functions arepreserved therein. Thus, the bispecific antibody binds to any antigenrecognized thereby at the same time with binding to FcγR and exhibitsADCC activity against cells expressing the antigen.

Provided that all the antigens recognized by the bispecific antibody areantigens specifically expressed in cancer, the bispecific antibodybinding to any of the antigens exhibits cytotoxic activity againstcancer cells and can therefore be expected to have a more efficientanticancer effect than that of the conventional antibody drug thatrecognizes one antigen. However, in the case where any one of theantigens recognized by the bispecific antibody is expressed in a normaltissue or is a cell expressed on immunocytes, damage on the normaltissue or release of cytokines occurs due to cross-linking with FcγR (J.Immunol. (1999) Aug. 1, 163 (3), 1246-52 (Non Patent Literature 15)). Asa result, strong adverse reactions are induced.

For example, catumaxomab is known as a bispecific antibody thatrecognizes a protein expressed on T cells and a protein expressed oncancer cells (cancer antigen). Catumaxomab binds, at two Fabs, thecancer antigen (EpCAM) and a CD3ε chain expressed on T cells,respectively. Catumaxomab induces T cell-mediated cytotoxic activitythrough binding to the cancer antigen and the CD3ε at the same time andinduces NK cell- or antigen-presenting cell (e.g., macrophage)-mediatedcytotoxic activity through binding to the cancer antigen and FcγR at thesame time. By use of these two cytotoxic activities, catumaxomabexhibits a high therapeutic effect on malignant ascites byintraperitoneal administration and has thus been approved in Europe(Cancer Treat Rev. (2010) Oct. 36 (6), 458-67 (Non Patent Literature16)). In addition, the administration of catumaxomab reportedly yieldscancer cell-reactive antibodies in some cases, demonstrating thatacquired immunity is induced (Future Oncol. (2012) Jan. 8 (1), 73-85(Non Patent Literature 17)). From this result, such antibodies havingboth of T cell-mediated cytotoxic activity and the effect brought aboutby cells such as NK cells or macrophages via FcγR (these antibodies areparticularly referred to as trifunctional antibodies) have receivedattention because a strong antitumor effect and induction of acquiredimmunity can be expected.

The trifunctional antibodies, however, bind to CD3ε and FcγR at the sametime even in the absence of a cancer antigen and therefore cross-linkCD3ε-expressing T cells to FcγR-expressing cells even in a cancercell-free environment to produce various cytokines in large amounts.Such cancer antigen-independent induction of production of variouscytokines restricts the current administration of the trifunctionalantibodies to an intraperitoneal route (Cancer Treat Rev. 2010 Oct. 36(6), 458-67 (Non Patent Literature 16)). The trifunctional antibodiesare very difficult to administer systemically due to serious cytokinestorm-like adverse reactions (Cancer Immunol Immunother. 2007 September;56 (9): 1397-406 (Non Patent Literature 18)).

The bispecific antibody of the conventional technique is capable ofbinding to both antigens, i.e., a first antigen cancer antigen (EpCAM)and a second antigen CD3ε, at the same time with binding to FcγR, andtherefore, cannot circumvent, in view of its molecular structure, suchadverse reactions caused by the binding to FcγR and the second antigenCD3ε at the same time.

In recent years, a modified antibody that causes cytotoxic activitymediated by T cells while circumventing adverse reactions has beenprovided by use of an Fc region having reduced binding activity againstFcγR (WO2012/073985).

Even such an antibody, however, fails to act on two immunoreceptors,i.e., CD3ε and FcγR, while binding to the cancer antigen, in view of itsmolecular structure.

An antibody that exerts both of cytotoxic activity mediated by T cellsand cytotoxic activity mediated by cells other than the T cells in acancer antigen-specific manner while circumventing adverse reactions hasnot yet been known.

CITATION LIST Patent Literature

-   Patent Literature 1: WO2000/042072-   Patent Literature 2: WO2006/019447

Non Patent Literature

-   Non Patent Literature 1: Nat. Biotechnol. (2005) 23, 1073-1078-   Non Patent Literature 2: Eur J Pharm Biopharm. (2005) 59 (3),    389-396-   Non Patent Literature 3: Immunol. Lett. (2002) 82, 57-65-   Non Patent Literature 4: Nat. Rev. Immunol. (2008) 8, 34-47-   Non Patent Literature 5: Ann. Rev. Immunol. (1988). 6. 251-81-   Non Patent Literature 6: Chem. Immunol. (1997), 65, 88-110-   Non Patent Literature 7: Eur. J. Immunol. (1993) 23, 1098-1104-   Non Patent Literature 8: Immunol. (1995) 86, 319-324-   Non Patent Literature 9: Proc. Natl. Acad. Sci. U.S.A. (2006) 103,    4005-4010-   Non Patent Literature 10: J. Biol. Chem. (2003) 278, 3466-3473-   Non Patent Literature 11: Nat. Biotech., (2011) 28, 502-10-   Non Patent Literature 12: Endocr Relat Cancer (2006) 13, 45-51-   Non Patent Literature 13: Nat. Rev. (2010), 10, 301-316-   Non Patent Literature 14: Peds (2010), 23 (4), 289-297-   Non Patent Literature 15: J. Immunol. (1999) Aug. 1, 163 (3),    1246-52-   Non Patent Literature 16: Cancer Treat Rev. (2010) Oct. 36 (6),    458-67-   Non Patent Literature 17: Future Oncol. (2012) Jan. 8 (1), 73-85-   Non Patent Literature 18: Cancer Immunol Immunother. 2007 September;    56 (9): 1397-406

SUMMARY OF INVENTION Technical Problem

The present invention has been made in light of these circumstances. Anobject of the present invention is to provide an antigen-bindingmolecule comprising: an antibody variable region that has bindingactivity against two different antigens (first antigen and secondantigen), but does not bind to these antigens at the same time; and avariable region binding to an antigen (third antigen) different fromthese antigens, a pharmaceutical composition comprising theantigen-binding molecule, and a method for producing the antigen-bindingmolecule.

Solution to Problem

The present inventors have conducted diligent studies to attain theobject. As a result, the present inventors have successfully prepared anantigen-binding molecule comprising: an antibody variable region thathas binding activity against two different antigens (first antigen andsecond antigen), but does not bind to these antigens at the same time;and a variable region binding to an antigen (third antigen) differentfrom these antigens, and enhanced activity brought about by thisantigen-binding molecule through the use of the binding activity of theantigen-binding molecule against the three different antigens. Inaddition, the present inventors have successfully prepared anantigen-binding molecule capable of circumventing the cross-linkingbetween different cells resulting from the binding of a conventionalmultispecific antigen-binding molecule to antigens expressed on thedifferent cells, which is considered to be responsible for adversereactions when the multispecific antigen-binding molecule is used as adrug.

More specifically, the present invention relates to the following:

[1] An antigen-binding molecule comprising:

an antibody variable region that is capable of binding to a firstantigen and a second antigen different from the first antigen, but doesnot bind to the first antigen and the second antigen at the same time;and a variable region binding to a third antigen different from thefirst antigen and the second antigen.

[2] An antigen-binding molecule comprising an antibody variable regionwith an amino acid altered in a heavy chain variable domain such thatthe variable region is capable of binding to a first antigen and asecond antigen different from the first antigen, but does not bind tothe first antigen and the second antigen at the same time.[3] The antigen-binding molecule according to [1] or [2], wherein thevariable region that does not bind to the first antigen and the secondantigen at the same time is a variable region that does not bind to thefirst antigen and the second antigen each expressed on a different cell,at the same time.[4] The antigen-binding molecule according to any of [1] to [3], furthercomprising an antibody Fc region.[5] The antigen-binding molecule according to [4], wherein the Fc regionis an Fc region having reduced binding activity against FcγR as comparedwith the Fc region of a naturally occurring human IgG1 antibody.[6] The antigen-binding molecule according to any of [1] to [5], whereinthe antigen-binding molecule is a multispecific antibody.[7] The antigen-binding molecule according to any of [1] to [6], whereinthe antibody variable region capable of binding to the first antigen andthe second antigen is a variable region having alteration of at leastone amino acid.[8] The antigen-binding molecule according to [7], wherein thealteration is substitution or insertion of at least one amino acid.[9] The antigen-binding molecule according to [7] or [8], wherein thealteration is substitution of a portion of the amino acid sequence of avariable region binding to the first antigen by an amino acid sequencebinding to the second antigen, or insertion of an amino acid sequencebinding to the second antigen to the amino acid sequence of a variableregion binding to the first antigen.[10] The antigen-binding molecule according to [8] or [9], wherein thenumber of amino acids to be inserted is 1 to 25.[11] The antigen-binding molecule according to any of [7] to [10],wherein the amino acid to be altered is an amino acid in a CDR1, CDR2,CDR3, or FR3 region of the antibody variable region.[12] The antigen-binding molecule according to any of [7] to [11],wherein the amino acid to be altered is an amino acid in a loop.[13] The antigen-binding molecule according to any of [7] to [11],wherein the amino acid to be altered is at least one amino acid selectedfrom Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to102 in an antibody H chain variable domain, and Kabat numberingpositions 24 to 34, 50 to 56, and 89 to 97 in an L chain variabledomain.[14] The antigen-binding molecule according to any of [1] to [13],wherein any one of the first antigen and the second antigen is amolecule specifically expressed on the surface of a T cell, and theother antigen is a molecule expressed on the surface of a T cell or anyother immunocyte.[15] The antigen-binding molecule according to [14], wherein any one ofthe first antigen and the second antigen is CD3, and the other antigenis FcγR, TLR, lectin, IgA, an immune checkpoint molecule, a TNFsuperfamily molecule, a TNFR superfamily molecule, or an NK receptormolecule.[16] The antigen-binding molecule according to [14] or [15], wherein thethird antigen is a molecule specifically expressed in a cancer tissue.[17] A pharmaceutical composition comprising an antigen-binding moleculeaccording to any of [1] to [16] and a pharmaceutically acceptablecarrier.[18] A method for producing an antigen-binding molecule according to anyof [1] to [16], the method comprising steps (i) to (iv):(i) preparing a library of antigen-binding molecules with at least oneamino acid altered in their antibody variable regions each binding tothe first antigen or the second antigen, wherein the altered variableregions differ in at least one amino acid from each other;(ii) selecting, from the prepared library, an antigen-binding moleculecomprising a variable region that has binding activity against the firstantigen and the second antigen, but does not bind to the first antigenand the second antigen at the same time;(iii) culturing a host cell comprising a nucleic acid encoding thevariable region of the antigen-binding molecule selected in the step(ii), and/or a nucleic acid encoding a variable region of anantigen-binding molecule binding to the third antigen, to express anantigen-binding molecule comprising the antibody variable region that iscapable of binding to the first antigen and the second antigen, but doesnot bind to the first antigen and the second antigen at the same time,and/or the variable region binding to the third antigen; and(iv) recovering the antigen-binding molecule from the host cellcultures.[19] The method according to [18], wherein the variable region that doesnot bind to the first antigen and the second antigen at the same time,contained in the antigen-binding molecule selected in the step (ii) is avariable region that does not bind to the first antigen and the secondantigen each expressed on a different cell, at the same time.[20] The method according to [18] or [19], wherein the host cellcultured in the step (iii) further comprises a nucleic acid encoding anantibody Fc region.[21] The method according to [20], wherein the Fc region is an Fc regionhaving reduced binding activity against FcγR as compared with the Fcregion of a naturally occurring human IgG1 antibody.[22] The method according to any of [18] to [21], wherein theantigen-binding molecule to be produced is a multispecific antibody.[23] The method according to any of [18] to [22], wherein the at leastone amino acid altered in the variable regions in the step (i) is asubstituted or inserted amino acid.[24] The method according to [23], wherein the number of inserted aminoacids is 1 to 25.[25] The method according to any of [18] to [24], wherein the alterationis alteration of an amino acid in a CDR1, CDR2, CDR3, or FR3 region ofthe antibody variable region.[26] The method according to any of [18] to [25], wherein the alterationis alteration of an amino acid in a loop.[27] The method according to any of [18] to [25], wherein the alterationis alteration of at least one amino acid selected from Kabat numberingpositions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in an antibody Hchain variable domain, and Kabat numbering positions 24 to 34, 50 to 56,and 89 to 97 in an L chain variable domain.[28] The method according to any of [18] to [27], wherein any one of thefirst antigen and the second antigen is a molecule specificallyexpressed on the surface of a T cell, and the other antigen is amolecule expressed on the surface of a T cell or any other immunocyte.[29] The method according to [28], wherein any one of the first antigenand the second antigen is CD3, and the other antigen is FcγR, TLR, IgA,lectin, an immune checkpoint molecule, a TNF superfamily molecule, aTNFR superfamily molecule, or an NK receptor molecule.[30] The method according to [28] or [29], wherein the third antigen isa molecule specifically expressed in a cancer tissue.[31] A method for treating cancer, comprising the step of administeringan antigen-binding molecule according to any of [1] to [16].[32] The antigen-binding molecule according to any of [1] to [16] foruse in the treatment of cancer.[33] Use of an antigen-binding molecule according to any of [1] to [16]in the production of a therapeutic agent for cancer.[34] A process for producing a therapeutic agent for cancer, comprisingthe step of using an antigen-binding molecule according to any of [1] to[16].[35] Those skilled in the art should understand that one of or anycombination of two or more of the aspects described above is alsoincluded in the present invention unless a technical contradictionarises on the basis of the technical common sense of those skilled inthe art.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptual diagram of an antibody that binds to a firstantigen and a second antigen, but does not bind to these antigens at thesame time.

FIG. 2 is a conceptual diagram of an antibody that does not causecross-linking because the antibody does not bind to two antigens at thesame time.

FIG. 3 is a conceptual diagram of an antibody that binds to two antigensat the same time, but does not link two cells at the same time.

FIG. 4 is a conceptual diagram of an antibody that cross-links a cancercell to a T cell expressing a first receptor.

FIG. 5 is a conceptual diagram of an antibody that cross-links a cancercell to a cell expressing a second receptor.

FIG. 6 is a conceptual diagram of an antibody that cross-links a cancercell to an immunocyte, but does not cross-link immunocytes.

FIG. 7 is a graph showing results of cell-ELISA of CE115 for CD3ε.

FIG. 8 is a diagram showing the molecular form of EGFR_ERY22_CE115.

FIG. 9 is a graph showing results of TDCC (SK-pca13a) ofEGFR_ERY22_CE115.

FIG. 10 is a graph showing the binding activity of humanized CE115against CD3ε.

FIG. 11 is a graph showing results of ECL-ELISA for detecting thebinding of RGD-inserted CE115 to integrin.

FIG. 12 is a graph showing results of ECL-ELISA for detecting thebinding of RGD-inserted CE115 to CD3ε.

FIG. 13 is a graph showing results of ECL-ELISA for detecting thebinding of RGD-inserted CE115 to integrin and CD3ε at the same time. Theresults about altered forms binding thereto at the same time are shown.

FIG. 14 is a graph showing results of ECL-ELISA for detecting thebinding of RGD-inserted CE115 to the antigens at the same time. Theresults about altered forms that do not bind to the antigens at the sametime are shown.

FIG. 15 is a graph showing results of ECL-ELISA for detecting thebinding of TLR2-binding peptide-inserted CE115 to TLR2.

FIG. 16 is a graph showing results of ECL-ELISA for detecting thebinding of TLR2-binding peptide-inserted CE115 to CD3ε.

FIG. 17 is a graph showing results of ECL-ELISA for detecting thebinding of TLR2-binding peptide-inserted CE115 to TLR2 and CD3 at thesame time.

FIG. 18 is an exemplary sensorgram of an antibody having a ratio of theamounts bound of less than 0.8. The ordinate depicts an RU value(response). The abscissa depicts time.

FIG. 19 is a diagram showing the binding of a Fab domain displayed by aphage to CD3ε and IL6R.

FIG. 20 is a diagram showing the binding of a Fab domain displayed by aphage to CD3ε and human IgA (hIgA). NC denotes its binding to anantigen-unimmobilized plate.

FIG. 21 is a diagram showing the binding of a clone converted to IgG toCD3ε and human IgA (hIgA).

FIG. 22 is a diagram showing that the binding of a clone converted toIgG to human IgA is inhibited by CD3ε so that the clone cannot bind tohuman IgA (hIgA) and CD3ε at the same time.

FIG. 23 is a diagram showing the binding of a Fab domain displayed by aphage to CD3ε and human CD154. NC denotes its binding to anantigen-unimmobilized plate.

FIG. 24 is a diagram showing the binding of a clone converted to IgG toCD3ε and human CD154.

FIG. 25 is a diagram showing that the binding of a clone converted toIgG to human CD154 is inhibited by CD3ε so that the clone cannot bind tohuman CD154 and CD3ε at the same time.

DESCRIPTION OF EMBODIMENTS

In the present invention, the “antibody variable region” usually means aregion comprising a domain constituted by four framework regions (FRs)and three complementarity-determining regions (CDRs) flanked thereby,and also includes a partial sequence thereof as long as the partialsequence has the activity of binding to a portion or the whole of anantigen. Particularly, a region comprising an antibody light chainvariable domain (VL) and an antibody heavy chain variable domain (VH) ispreferred. The antibody variable region of the present invention mayhave an arbitrary sequence and may be a variable region derived from anyantibody such as a mouse antibody, a rat antibody, a rabbit antibody, agoat antibody, a camel antibody, and a humanized antibody obtained bythe humanization of any of these nonhuman antibodies, and a humanantibody. The “humanized antibody”, also called reshaped human antibody,is obtained by grafting complementarity determining regions (CDRs) of anon-human mammal-derived antibody, for example, a mouse antibody tohuman antibody CDRs. Methods for identifying CDRs are known in the art(Kabat et al., Sequence of Proteins of Immunological Interest (1987),National Institute of Health, Bethesda, Md.; and Chothia et al., Nature(1989) 342: 877). General gene recombination approaches therefor arealso known in the art (see European Patent Application Publication No.EP 125023 and WO 96/02576).

The “antibody variable region” of the present invention that does “notbind to the first antigen and the second antigen at the same time” meansthat the antibody variable region of the present invention cannot bindto the second antigen in a state bound with the first antigen whereasthe variable region cannot bind to the first antigen in a state boundwith the second antigen. In this context, the phrase “not bind to thefirst antigen and the second antigen at the same time” also includes notcross-linking a cell expressing the first antigen to a cell expressingthe second antigen, or not binding to the first antigen and the secondantigen each expressed on a different cell, at the same time. Thisphrase further includes the case where the variable region is capable ofbinding to both the first antigen and the second antigen at the sametime when the first antigen and the second antigen are not expressed oncell membranes, as with soluble proteins, or both reside on the samecell, but cannot bind to the first antigen and the second antigen eachexpressed on a different cell, at the same time. Such an antibodyvariable region is not particularly limited as long as the antibodyvariable region has these functions. Examples thereof can includevariable regions derived from an IgG-type antibody variable region bythe alteration of a portion of its amino acids so as to bind to thedesired antigen. The amino acid to be altered is selected from, forexample, amino acids whose alteration does not cancel the binding to theantigen, in an antibody variable region binding to the first antigen orthe second antigen.

In this context, the phrase “expressed on different cells” merely meansthat the antigens are expressed on separate cells. The combination ofsuch cells may be, for example, the same types of cells such as a T celland another T cell, or may be different types of cells such as a T celland an NK cell.

In the present invention, one amino acid alteration may be used alone,or a plurality of amino acid alterations may be used in combination.

In the case of using a plurality of amino acid alterations incombination, the number of the alterations to be combined is notparticularly limited and can be appropriately set within a range thatcan attain the object of the invention. The number of the alterations tobe combined is, for example, 2 or more and 30 or less, preferably 2 ormore and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less,2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 orless, or 2 or more and 3 or less.

The plurality of amino acid alterations to be combined may be added toonly the antibody heavy chain variable domain or light chain variabledomain or may be appropriately distributed to both of the heavy chainvariable domain and the light chain variable domain.

One or more amino acid residues in the variable region are acceptable asthe amino acid residue to be altered as long as the antigen-bindingactivity is maintained. In the case of altering an amino acid in thevariable region, the resulting variable region preferably maintains thebinding activity of the corresponding unaltered antibody and preferablyhas, for example, 50% or higher, more preferably 80% or higher, furtherpreferably 100% or higher, of the binding activity before thealteration, though the variable region according to the presentinvention is not limited thereto. The binding activity may be increasedby the amino acid alteration and may be, for example, 2 times, 5 times,or 10 times the binding activity before the alteration.

Examples of the region preferred for the amino acid alteration includesolvent-exposed regions and loops in the variable region. Among others,CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabatnumbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the Hchain variable domain and Kabat numbering positions 24 to 34, 50 to 56,and 89 to 97 in the L chain variable domain are preferred. Kabatnumbering positions 31, 52a to 61, 71 to 74, and 97 to 101 in the Hchain variable domain and Kabat numbering positions 24 to 34, 51 to 56,and 89 to 96 in the L chain variable domain are more preferred. Also, anamino acid that increases antigen-binding activity may be furtherintroduced at the time of the amino acid alteration.

In the present invention, the “loop” means a region containing residuesthat are not involved in the maintenance of an immunoglobulin β barrelstructure.

In the present invention, the amino acid alteration means substitution,deletion, addition, insertion, or modification, or a combinationthereof. In the present invention, the amino acid alteration can be usedinterchangeably with amino acid mutation and used in the same sensetherewith.

The substitution of an amino acid residue is carried out by replacementwith another amino acid residue for the purpose of altering, forexample, any of the following (a) to (c): (a) the polypeptide backbonestructure of a region having a sheet structure or helix structure; (b)the electric charge or hydrophobicity of a target site; and (c) the sizeof a side chain.

Amino acid residues are classified into the following groups on thebasis of general side chain properties: (1) hydrophobic residues:norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral hydrophilicresidues: Cys, Ser, Thr, Asn, and Gln; (3) acidic residues: Asp and Glu;(4) basic residues: His, Lys, and Arg; (5) residues that influence chainorientation: Gly and Pro; and (6) aromatic residues: Trp, Tyr, and Phe.

The substitution of amino acid residues within each of these groups iscalled conservative substitution, while the substitution of an aminoacid residue in one of these groups by an amino acid residue in anothergroup is called non-conservative substitution.

The substitution according to the present invention may be theconservative substitution or may be the non-conservative substitution.Alternatively, the conservative substitution and the non-conservativesubstitution may be combined.

The alteration of an amino acid residue also includes: the selection ofa variable region that is capable of binding to the first antigen andthe second antigen, but cannot bind to these antigens at the same time,from those obtained by the random alteration of amino acids whosealteration does not cancel the binding to the antigen, in the antibodyvariable region binding to the first antigen or the second antigen; andalteration to insert a peptide previously known to have binding activityagainst the desired antigen, to the region mentioned above. Examples ofthe peptide previously known to have binding activity against thedesired antigen include peptides shown in Table 1.

TABLE 1 Binding partner/ protein of interest References VEGFR J BiolChem. 2002 Nov. 8; 277(45): 43137-42. Epub 2002 Aug. 14., EMBO J. 2000Apr. 3; 19(7): 1525-33., J Med Chem. 2010 Jun. 10; 53(11): 4428-40. TNFRMol Immunol. 2004 Jul.; 41(8): 741-9., Eur J Pharmacol. 2011 Apr. 10;656(1-3): 119-24. TLR5 J Immunol 2010; 185; 1744-1754 TLR4 PLoS ONE,Feb. 2012 | Volume 7 | Issue 2 | e30839 TLR2 WO2006/083706A2, T cell VLAreceptor Int Immunopharmacol. 2003 Mar.; 3(3): 435-43. PDGFR BiochemicalPharmacology(2003), 66(7), 1307-1317, FEBS Lett. 1997 Dec. 15; 419(2-3):166-70. Naip5(NLR) NATURE IMMUNOLOGY VOLUME 9 NUMBER 10 OCT. 2008 1171-Integrin WO 95/14714, WO 97/08203, WO 98/10795, WO 99/24462, J. Biol.Chem. 274: 1979-1985 FcgRIIa J Biol Chem. 2009 Jan. 9; 284(2): 1126-35EGFR Journal of Biotechnology(2005), 116(3) 211-219 DR5 agonist Journalof Biotechnology(2006), 361(3) 522-536 CXCR4 Science 330, 1066 (2010);Vol. 330 no. 6007 pp. 1066-1071 CD40 Eur J Biochem. 2003 May; 270(10):2287-94. CD154 J Mol Med (Berl). 2009 Feb.; 87(2): 181-97.

According to one aspect, the present invention provides anantigen-binding molecule comprising an antibody variable region with anamino acid altered in a heavy chain variable domain such that thevariable region is capable of binding to a first antigen and a secondantigen different from the first antigen, but does not bind to the firstantigen and the second antigen at the same time. The antibody variableregion that is capable of binding to a first antigen and a secondantigen different from the first antigen, but does not bind to the firstantigen and the second antigen at the same time can be prepared, forexample, by introducing the amino acid alteration (substitution,deletion, addition, insertion, or modification, or a combinationthereof) mentioned above to a heavy chain variable domain. The positionto which the amino acid alteration is introduced is preferably a heavychain variable domain. Examples of a more preferred region includesolvent-exposed regions and loops in the variable domain. Among others,CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabatnumbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the Hchain variable domain are preferred. Kabat numbering positions 31, 52ato 61, 71 to 74, and 97 to 101 in the H chain variable domain are morepreferred. Also, an amino acid that increases antigen-binding activitymay be further introduced at the time of the amino acid alteration.

In the antibody variable region of the present invention, the alterationmentioned above may be combined with alteration known in the art. Forexample, the modification of N-terminal glutamine of the variable regionto pyroglutamic acid by pyroglutamylation is a modification well knownto those skilled in the art. Thus, the antibody of the present inventionhaving glutamine at the N terminus of its heavy chain may contain avariable region with this N-terminal glutamine modified to pyroglutamicacid.

Such an antibody variable region may further have amino acid alterationto improve, for example, antigen binding, pharmacokinetics, stability,or antigenicity. The antibody variable region of the present inventionmay be altered so as to have pH dependent binding activity against anantigen and be thereby capable of repetitively binding to the antigen(WO2009/125825).

Also, amino acid alteration to change antigen-binding activity accordingto the concentration of a target tissue-specific compound may be addedto, for example, such an antibody variable region binding to a thirdantigen (WO2013/180200).

The variable region may be further altered for the purpose of, forexample, enhancing binding activity, improving specificity, reducing pI,conferring pH-dependent antigen-binding properties, improving thethermal stability of binding, improving solubility, improving stabilityagainst chemical modification, improving heterogeneity derived from asugar chain, avoiding a T cell epitope identified by use of in silicoprediction or in vitro T cell-based assay for reduction inimmunogenicity, or introducing a T cell epitope for activatingregulatory T cells (mAbs 3: 243-247, 2011).

Whether the antibody variable region of the present invention is“capable of binding to the first antigen and the second antigen” can bedetermined by a method known in the art.

This can be determined by, for example, an electrochemiluminescencemethod (ECL method) (BMC Research Notes 2011, 4: 281).

Specifically, for example, a low-molecular antibody composed of a regioncapable of binding to the first antigen and the second antigen, forexample, a Fab region, of a biotin-labeled antigen-binding molecule tobe tested, or a monovalent antibody (antibody lacking one of the two Fabregions carried by a usual antibody) thereof is mixed with the firstantigen or the second antigen labeled with sulfo-tag (Ru complex), andthe mixture is added onto a streptavidin-immobilized plate. In thisoperation, the biotin-labeled antigen-binding molecule to be testedbinds to streptavidin on the plate. Light is developed from thesulfo-tag, and the luminescence signal can be detected using SectorImager 600 or 2400 (MSD K.K.) or the like to thereby confirm the bindingof the aforementioned region of the antigen-binding molecule to betested to the first antigen or the second antigen.

Alternatively, this assay may be conducted by ELISA, FACS (fluorescenceactivated cell sorting), ALPHAScreen (amplified luminescent proximityhomogeneous assay screen), the BIACORE method based on a surface plasmonresonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

Specifically, the assay can be conducted using, for example, aninteraction analyzer Biacore (GE Healthcare Japan Corp.) based on asurface plasmon resonance (SPR) phenomenon. The Biacore analyzerincludes any model such as Biacore T100, T200, X100, A100, 4000, 3000,2000, 1000, or C. Any sensor chip for Biacore, such as a CM7, CM5, CM4,CM3, C1, SA, NTA, L1, HPA, or Au chip, can be used as a sensor chip.Proteins for capturing the antigen-binding molecule of the presentinvention, such as protein A, protein G, protein L, anti-human IgGantibodies, anti-human IgG-Fab, anti-human L chain antibodies,anti-human Fc antibodies, antigenic proteins, or antigenic peptides, areimmobilized onto the sensor chip by a coupling method such as aminecoupling, disulfide coupling, or aldehyde coupling. The first antigen orthe second antigen is injected thereon as an analyte, and theinteraction is measured to obtain a sensorgram. In this operation, theconcentration of the first antigen or the second antigen can be selectedwithin the range of a few μM to a few pM according to the interactionstrength (e.g., KD) of the assay sample.

Alternatively, the first antigen or the second antigen may beimmobilized instead of the antigen-binding molecule onto the sensorchip, with which the antibody sample to be evaluated is in turn allowedto interact. Whether the antibody variable region of the antigen-bindingmolecule of the present invention has binding activity against the firstantigen or the second antigen can be confirmed on the basis of adissociation constant (KD) value calculated from the sensorgram of theinteraction or on the basis of the degree of increase in the sensorgramafter the action of the antigen-binding molecule sample over the levelbefore the action.

The ALPHAScreen is carried out by the ALPHA technology using two typesof beads (donor and acceptor) on the basis of the following principle:luminescence signals are detected only when these two beads are locatedin proximity through the biological interaction between a molecule boundwith the donor bead and a molecule bound with the acceptor bead. Alaser-excited photosensitizer in the donor bead converts ambient oxygento singlet oxygen having an excited state. The singlet oxygen diffusesaround the donor bead and reaches the acceptor bead located in proximitythereto to thereby cause chemiluminescent reaction in the bead, whichfinally emits light. In the absence of the interaction between themolecule bound with the donor bead and the molecule bound with theacceptor bead, singlet oxygen produced by the donor bead does not reachthe acceptor bead. Thus, no chemiluminescent reaction occurs.

One (ligand) of the substances between which the interaction is to beobserved is immobilized onto a thin gold film of a sensor chip. Thesensor chip is irradiated with light from the back such that totalreflection occurs at the interface between the thin gold film and glass.As a result, a site having a drop in reflection intensity (SPR signal)is formed in a portion of reflected light. The other (analyte) of thesubstances between which the interaction is to be observed is injectedon the surface of the sensor chip. Upon binding of the analyte to theligand, the mass of the immobilized ligand molecule is increased tochange the refractive index of the solvent on the sensor chip surface.This change in the refractive index shifts the position of the SPRsignal (on the contrary, the dissociation of the bound molecules getsthe signal back to the original position). The Biacore system plots onthe ordinate the amount of the shift, i.e., change in mass on the sensorchip surface, and displays time-dependent change in mass as assay data(sensorgram). The amount of the analyte bound to the ligand captured onthe sensor chip surface (amount of change in response on the sensorgrambetween before and after the interaction of the analyte) can bedetermined from the sensorgram. However, since the amount bound alsodepends on the amount of the ligand, the comparison must be performedunder conditions where substantially the same amounts of the ligand areused. Kinetics, i.e., an association rate constant (ka) and adissociation rate constant (kd), can be determined from the curve of thesensorgram, while affinity (KD) can be determined from the ratio betweenthese constants. Inhibition assay is also preferably used in the BIACOREmethod. Examples of the inhibition assay are described in Proc. Natl.Acad. Sci. USA (2006) 103 (11), 4005-4010.

Whether the antigen-binding molecule of the present invention does “notbind to the first antigen and the second antigen at the same time” canbe confirmed by: confirming the antigen-binding molecule to have bindingactivity against the first antigen and the second antigen; then allowingeither of the first antigen or the second antigen to bind in advance tothe antigen-binding molecule comprising the variable region having thisbinding activity; and then determining the presence or absence of itsbinding activity against the other antigen by the method mentionedabove.

Alternatively, this can also be confirmed by determining whether thebinding of the antigen-binding molecule to either of the first antigenor the second antigen immobilized on an ELISA plate or a sensor chip isinhibited by the addition of the other antigen into the solution.

Specifically, in the case of using, for example, the ECL method, abiotin-labeled antigen-binding molecule to be tested, the first antigenlabeled with sulfo-tag (Ru complex), and an unlabeled second antigen areprepared. When the antigen-binding molecule to be tested is capable ofbinding to the first antigen and the second antigen, but does not bindto the first antigen and the second antigen at the same time, theluminescence signal of the sulfo-tag is detected in the absence of theunlabeled second antigen by adding the mixture of the antigen-bindingmolecule to be tested and the first antigen onto astreptavidin-immobilized plate, followed by light development. Bycontrast, the luminescence signal is decreased in the presence of thesecond antigen. This decrease in luminescence signal can be quantifiedto determine relative binding activity.

In the case of the ALPHAScreen, the antigen-binding molecule to betested interacts with the first antigen in the absence of the competingsecond antigen to generate signals of 520 to 620 nm. The untagged secondantigen competes with the first antigen for the interaction with theantigen-binding molecule to be tested. Decrease in fluorescence causedas a result of the competition can be quantified to thereby determinerelative binding activity. The polypeptide biotinylation usingsulfo-NHS-biotin or the like is known in the art. The first antigen canbe tagged with GST by an appropriately adopted method which involves,for example: fusing a polynucleotide encoding the first antigen in flamewith a polynucleotide encoding GST; and allowing the resulting fusiongene to be expressed by cells or the like harboring vectors capable ofexpression thereof, followed by purification using a glutathione column.The obtained signals are preferably analyzed using, for example,software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) adapted toa one-site competition model based on nonlinear regression analysis.This analysis may be similarly conducted using the tagged second antigenand the untagged first antigen.

Alternatively, a method using fluorescence resonance energy transfer(FRET) may be used. FRET is a phenomenon in which excitation energy istransferred directly between two fluorescent molecules located inproximity to each other by electron resonance. When FRET occurs, theexcitation energy of a donor (fluorescent molecule having an excitedstate) is transferred to an acceptor (another fluorescent moleculelocated near the donor) so that the fluorescence emitted from the donordisappears (to be precise, the lifetime of the fluorescence isshortened) and instead, the fluorescence is emitted from the acceptor.By use of this phenomenon, whether or not to be dual-Fab can beanalyzed. For example, when the first antigen carrying a fluorescencedonor and the second antigen carrying a fluorescence acceptor bind tothe antigen-binding molecule to be tested at the same time, thefluorescence of the donor disappears while the fluorescence is emittedfrom the acceptor. Therefore, change in fluorescence wavelength isobserved. Such an antibody is confirmed not to be dual-Fab. On the otherhand, if the mixing of the first antigen, the second antigen, and theantigen-binding molecule to be tested does not change the fluorescencewavelength of the fluorescence donor bound with the first antigen, thisantigen-binding molecule to be tested can be regarded as dual-Fab.

For example, a biotin-labeled antigen-binding molecule to be tested isallowed to bind to streptavidin on the donor bead, while the firstantigen tagged with glutathione S transferase (GST) is allowed to bindto the acceptor bead. The antigen-binding molecule to be testedinteracts with the first antigen in the absence of the competing secondantigen to generate signals of 520 to 620 nm. The untagged secondantigen competes with the first antigen for the interaction with theantigen-binding molecule to be tested. Decrease in fluorescence causedas a result of the competition can be quantified to thereby determinerelative binding activity. The polypeptide biotinylation usingsulfo-NHS-biotin or the like is known in the art. The first antigen canbe tagged with GST by an appropriately adopted method which involves,for example: fusing a polynucleotide encoding the first antigen in flamewith a polynucleotide encoding GST; and allowing the resulting fusiongene to be expressed by cells or the like harboring vectors capable ofexpression thereof, followed by purification using a glutathione column.The obtained signals are preferably analyzed using, for example,software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) adapted toa one-site competition model based on nonlinear regression analysis.

The tagging is not limited to the GST tagging and may be carried outwith any tag such as, but not limited to, a histidine tag, MBP, CBP, aFlag tag, an HA tag, a V5 tag, or a c-myc tag. The binding of theantigen-binding molecule to be tested to the donor bead is not limitedto the binding using biotin-streptavidin reaction. Particularly, whenthe antigen-binding molecule to be tested comprises Fc, a possiblemethod involves allowing the antigen-binding molecule to be tested tobind via an Fc-recognizing protein such as protein A or protein G on thedonor bead.

Also, the case where the variable region is capable of binding to thefirst antigen and the second antigen at the same time when the firstantigen and the second antigen are not expressed on cell membranes, aswith soluble proteins, or both reside on the same cell, but cannot bindto the first antigen and the second antigen each expressed on adifferent cell, at the same time can also be assayed by a method knownin the art.

Specifically, the antigen-binding molecule to be tested has beenconfirmed to be positive in ECL-ELISA for detecting binding to the firstantigen and the second antigen at the same time is also mixed with acell expressing the first antigen and a cell expressing the secondantigen. The antigen-binding molecule to be tested can be shown to beincapable of binding to the first antigen and the second antigenexpressed on different cells, at the same time unless theantigen-binding molecule and these cells bind to each other at the sametime. This assay can be conducted by, for example, cell-based ECL-ELISA.The cell expressing the first antigen is immobilized onto a plate inadvance. After binding of the antigen-binding molecule to be testedthereto, the cell expressing the second antigen is added to the plate. Adifferent antigen expressed only on the cell expressing the secondantigen is detected using a sulfo-tag-labeled antibody against thisantigen. A signal is observed when the antigen-binding molecule binds tothe two antigens respectively expressed on the two cells, at the sametime. No signal is observed when the antigen-binding molecule does notbind to these antigens at the same time.

Alternatively, this assay may be conducted by the ALPHAScreen method.The antigen-binding molecule to be tested is mixed with a cellexpressing the first antigen bound with the donor bead and a cellexpressing the second antigen bound with the acceptor bead. A signal isobserved when the antigen-binding molecule binds to the two antigensexpressed on the two cells respectively, at the same time. No signal isobserved when the antigen-binding molecule does not bind to theseantigens at the same time.

Alternatively, this assay may also be conducted by an Octet interactionanalysis method. First, a cell expressing the first antigen tagged witha peptide tag is allowed to bind to a biosensor that recognizes thepeptide tag. A cell expressing the second antigen and theantigen-binding molecule to be tested are placed in wells and analyzedfor interaction. A large wavelength shift caused by the binding of theantigen-binding molecule to be tested and the cell expressing the secondantigen to the biosensor is observed when the antigen-binding moleculebinds to the two antigens expressed on the two cells respectively, atthe same time. A small wavelength shift caused by the binding of onlythe antigen-binding molecule to be tested to the biosensor is observedwhen the antigen-binding molecule does not bind to these antigens at thesame time.

Instead of these methods based on the binding activity, assay based onbiological activity may be conducted. For example, a cell expressing thefirst antigen and a cell expressing the second antigen are mixed withthe antigen-binding molecule to be tested, and cultured. The twoantigens expressed on the two cells respectively are mutually activatedvia the antigen-binding molecule to be tested when the antigen-bindingmolecule binds to these two antigens at the same time. Therefore, changein activation signal, such as increase in the respective downstreamphosphorylation levels of the antigens, can be detected. Alternatively,cytokine production is induced as a result of the activation. Therefore,the amount of cytokines produced can be measured to thereby confirmwhether or not to bind to the two cells at the same time.

In the present invention, the “Fc region” refers to a region comprisinga fragment consisting of a hinge or a portion thereof and CH2 and CH3domains in an antibody molecule. The Fc region of IgG class means, butis not limited to, a region from, for example, cysteine 226 (EUnumbering (also referred to as EU index herein)) to the C terminus orproline 230 (EU numbering) to the C terminus. The Fc region can bepreferably obtained by the partial digestion of, for example, an IgG1,IgG2, IgG3, or IgG4 monoclonal antibody with a proteolytic enzyme suchas pepsin followed by the re-elution of a fraction adsorbed on a proteinA column or a protein G column. Such a proteolytic enzyme is notparticularly limited as long as the enzyme is capable of digesting awhole antibody to restrictively form Fab or F(ab′)2 under appropriatelyset reaction conditions (e.g., pH) of the enzyme. Examples thereof caninclude pepsin and papain.

In the present invention, the “antigen-binding molecule” is notparticularly limited as long as the molecule comprises the “antibodyvariable region” of the present invention. The antigen-binding moleculemay further comprise a peptide or a protein having a length ofapproximately 5 or more amino acids. The peptide or the protein is notlimited to a peptide or a protein derived from an organism, and may be,for example, a polypeptide consisting of an artificially designedsequence. Also, a natural polypeptide, a synthetic polypeptide, arecombinant polypeptide, or the like may be used.

Preferred examples of the antigen-binding molecule of the presentinvention can include an antigen-binding molecule comprising an antibodyFc region.

An Fc region derived from, for example, naturally occurring IgG can beused as the “Fc region” of the present invention. In this context, thenaturally occurring IgG means a polypeptide that contains an amino acidsequence identical to that of IgG found in nature and belongs to a classof an antibody substantially encoded by an immunoglobulin gamma gene.The naturally occurring human IgG means, for example, naturallyoccurring human IgG1, naturally occurring human IgG2, naturallyoccurring human IgG3, or naturally occurring human IgG4. The naturallyoccurring IgG also includes variants or the like spontaneously derivedtherefrom. A plurality of allotype sequences based on gene polymorphismare described as the constant regions of human IgG1, human IgG2, humanIgG3, and human IgG4 antibodies in Sequences of proteins ofimmunological interest, NIH Publication No. 91-3242, any of which can beused in the present invention. Particularly, the sequence of human IgG1may have DEL or EEM as an amino acid sequence of EU numbering positions356 to 358.

The antibody Fc region is found as, for example, an Fc region of IgA1,IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM type. For example, an Fcregion derived from a naturally occurring human IgG antibody can be usedas the antibody Fc region of the present invention. For example, an Fcregion derived from a constant region of naturally occurring IgG,specifically, a constant region (SEQ ID NO: 1) originated from naturallyoccurring human IgG1, a constant region (SEQ ID NO: 2) originated fromnaturally occurring human IgG2, a constant region (SEQ ID NO: 3)originated from naturally occurring human IgG3, or a constant region(SEQ ID NO: 4) originated from naturally occurring human IgG4 can beused as the Fc region of the present invention. The constant region ofnaturally occurring IgG also includes variants or the like spontaneouslyderived therefrom.

The Fc region of the present invention is particularly preferably an Fcregion having reduced binding activity against an Fcγ receptor. In thiscontext, the Fcγ receptor (also referred to as FcγR herein) refers to areceptor capable of binding to the Fc region of IgG1, IgG2, IgG3, orIgG4 and means any member of the protein family substantially encoded byFcγ receptor genes. In humans, this family includes, but is not limitedto: FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 (H type) andR131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2); and any yet-to-be-discovered human FcγR or FcγRisoform or allotype. The FcγR includes those derived from humans, mice,rats, rabbits, and monkeys. The FcγR is not limited to these moleculesand may be derived from any organism. The mouse FcγRs include, but arenot limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIII-2 (CD16-2), and any yet-to-be-discovered mouse FcγR or FcγRisoform or allotype. Preferred examples of such Fcγ receptors includehuman FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16),and/or FcγRIIIb (CD16).

The FcγR is found in the forms of an activating receptor having ITAM(immunoreceptor tyrosine-based activation motif) and an inhibitoryreceptor having ITIM (immunoreceptor tyrosine-based inhibitory motif).The FcγR is classified into activating FcγR (FcγRI, FcγRIIa R, FcγRIIaH, FcγRIIIa, and FcγRIIIb) and inhibitory FcγR (FcγRIIb).

The polynucleotide sequence and the amino acid sequence of FcγRI aredescribed in NM_000566.3 and NP_000557.1, respectively; thepolynucleotide sequence and the amino acid sequence of FcγRIIa aredescribed in BC020823.1 and AAH20823.1, respectively; the polynucleotidesequence and the amino acid sequence of FcγRIIb are described inBC146678.1 and AAI46679.1, respectively; the polynucleotide sequence andthe amino acid sequence of FcγRIIIa are described in BC033678.1 andAAH33678.1, respectively; and the polynucleotide sequence and the aminoacid sequence of FcγRIIIb are described in BC128562.1 and AAI28563.1,respectively (RefSeq registration numbers). FcγRIIa has two types ofgene polymorphisms that substitute the 131st amino acid of FcγRIIa byhistidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990).FcγRIIb has two types of gene polymorphisms that substitute the 232ndamino acid of FcγRIIb by isoleucine (I type) or threonine (T type)(Arthritis. Rheum. 46: 1242-1254 (2002)). FcγRIIIa has two types of genepolymorphisms that substitute the 158th amino acid of FcγRIIIa by valine(V type) or phenylalanine (F type) (J. Clin. Invest. 100 (5): 1059-1070(1997)). FcγRIIIb has two types of gene polymorphisms (NA1 type and NA2type) (J. Clin. Invest. 85: 1287-1295 (1990)).

The reduced binding activity against an Fcγ receptor can be confirmed bya well-known method such as FACS, ELISA format, ALPHAScreen (amplifiedluminescent proximity homogeneous assay screen), or the BIACORE methodbased on a surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad.Sci. USA (2006) 103 (11), 4005-4010).

The ALPHAScreen method is carried out by the ALPHA technology using twotypes of beads (donor and acceptor) on the basis of the followingprinciple: luminescence signals are detected only when these two beadsare located in proximity through the biological interaction between amolecule bound with the donor bead and a molecule bound with theacceptor bead. A laser-excited photosensitizer in the donor beadconverts ambient oxygen to singlet oxygen having an excited state. Thesinglet oxygen diffuses around the donor bead and reaches the acceptorbead located in proximity thereto to thereby cause chemiluminescentreaction in the bead, which finally emits light. In the absence of theinteraction between the molecule bound with the donor bead and themolecule bound with the acceptor bead, singlet oxygen produced by thedonor bead does not reach the acceptor bead. Thus, no chemiluminescentreaction occurs.

For example, a biotin-labeled antigen-binding molecule is allowed tobind to the donor bead, while a glutathione S transferase (GST)-taggedFcγ receptor is allowed to bind to the acceptor bead. In the absence ofa competing antigen-binding molecule having a mutated Fc region, anantigen-binding molecule having a wild-type Fc region interacts with theFcγ receptor to generate signals of 520 to 620 nm. The untaggedantigen-binding molecule having a mutated Fc region competes with theantigen-binding molecule having a wild-type Fc region for theinteraction with the Fcγ receptor. Decrease in fluorescence caused as aresult of the competition can be quantified to thereby determinerelative binding affinity. The antigen-binding molecule (e.g., antibody)biotinylation using sulfo-NHS-biotin or the like is known in the art.The Fcγ receptor can be tagged with GST by an appropriately adoptedmethod which involves, for example: fusing a polynucleotide encoding theFcγ receptor in flame with a polynucleotide encoding GST; and allowingthe resulting fusion gene to be expressed by cells or the like harboringvectors capable of expression thereof, followed by purification using aglutathione column. The obtained signals are preferably analyzed using,for example, software GRAPHPAD PRISM (GraphPad Software, Inc., SanDiego) adapted to a one-site competition model based on nonlinearregression analysis.

One (ligand) of the substances between which the interaction is to beobserved is immobilized onto a thin gold film of a sensor chip. Thesensor chip is irradiated with light from the back such that totalreflection occurs at the interface between the thin gold film and glass.As a result, a site having a drop in reflection intensity (SPR signal)is formed in a portion of reflected light. The other (analyte) of thesubstances between which the interaction is to be observed is injectedon the surface of the sensor chip. Upon binding of the analyte to theligand, the mass of the immobilized ligand molecule is increased tochange the refractive index of the solvent on the sensor chip surface.This change in the refractive index shifts the position of the SPRsignal (on the contrary, the dissociation of the bound molecules getsthe signal back to the original position). The Biacore system plots onthe ordinate the amount of the shift, i.e., change in mass on the sensorchip surface, and displays time-dependent change in mass as assay data(sensorgram). Kinetics, i.e., an association rate constant (ka) and adissociation rate constant (kd), can be determined from the curve of thesensorgram, while affinity (KD) can be determined from the ratio betweenthese constants. Inhibition assay is also preferably used in the BIACOREmethod. Examples of the inhibition assay are described in Proc. Natl.Acad. Sci. USA (2006) 103 (11), 4005-4010.

In the present specification, the reduced binding activity against anFcγ receptor means that the antigen-binding molecule to be testedexhibits binding activity of, for example, 50% or lower, preferably 45%or lower, 40% or lower, 35% or lower, 30% or lower, 20% or lower, or 15%or lower, particularly preferably 10% or lower, 9% or lower, 8% orlower, 7% or lower, 6% or lower, 5% or lower, 4% or lower, 3% or lower,2% or lower, or 1% or lower, compared with the binding activity of acontrol antigen-binding molecule comprising an Fc region on the basis ofthe analysis method described above.

An antigen-binding molecule having an IgG1, IgG2, IgG3, or IgG4monoclonal antibody Fc region can be appropriately used as the controlantigen-binding molecule. The structure of the Fc region is described inSEQ ID NO: 1 (RefSeq registration No. AAC82527.1 with A added to the Nterminus), SEQ ID NO: 2 (RefSeq registration No. AAB59393.1 with A addedto the N terminus), SEQ ID NO: 3 (RefSeq registration No. CAA27268.1with A added to the N terminus), or SEQ ID NO: 4 (RefSeq registrationNo. AAB59394.1 with A added to the N terminus). In the case of using anantigen-binding molecule having a variant of the Fc region of anantibody of a certain isotype as a test substance, an antigen-bindingmolecule having the Fc region of the antibody of this certain isotype isused as a control to test the effect of the mutation in the variant onthe binding activity against an Fcγ receptor. The antigen-bindingmolecule having the Fc region variant thus confirmed to have reducedbinding activity against an Fcγ receptor is appropriately prepared.

For example, a 231A-238S deletion (WO 2009/011941), C226S, C229S, P238S,(C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod.Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, or L235A(Blood (2007) 109, 1185-1192) (these amino acids are defined accordingto the EU numbering) variant is known in the art as such a variant.

Preferred examples thereof include antigen-binding molecules having anFc region derived from the Fc region of an antibody of a certain isotypeby the substitution of any of the following constituent amino acids:amino acids at positions 220, 226, 229, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298,299, 300, 325, 327, 328, 329, 330, 331, and 332 defined according to theEU numbering. The isotype of the antibody from which the Fc region isoriginated is not particularly limited, and an Fc region originated froman IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be appropriatelyused. An Fc region originated from a naturally occurring human IgG1antibody is preferably used.

For example, an antigen-binding molecule having an Fc region derivedfrom an IgG1 antibody Fc region by any of the following substitutiongroups of the constituent amino acids (the number represents theposition of an amino acid residue defined according to the EU numbering;the one-letter amino acid code positioned before the number representsan amino acid residue before the substitution; and the one-letter aminoacid code positioned after the number represents an amino acid residuebefore the substitution):

(a) L234F, L235E, and P331S, (b) C226S, C229S, and P238S, (c) C226S andC229S, and (d) C226S, C229S, E233P, L234V, and L235A

or by the deletion of an amino acid sequence from positions 231 to 238defined according to the EU numbering can also be appropriately used.

An antigen-binding molecule having an Fc region derived from an IgG2antibody Fc region by any of the following substitution groups of theconstituent amino acids (the number represents the position of an aminoacid residue defined according to the EU numbering; the one-letter aminoacid code positioned before the number represents an amino acid residuebefore the substitution; and the one-letter amino acid code positionedafter the number represents an amino acid residue before thesubstitution):

(e) H268Q, V309L, A330S, and P331S, (f) V234A, (g) G237A, (h) V234A andG237A, (i) A235E and G237A, and (j) V234A, A235E, and G237A

defined according to the EU numbering can also be appropriately used.

An antigen-binding molecule having an Fc region derived from an IgG3antibody Fc region by any of the following substitution groups of theconstituent amino acids (the number represents the position of an aminoacid residue defined according to the EU numbering; the one-letter aminoacid code positioned before the number represents an amino acid residuebefore the substitution; and the one-letter amino acid code positionedafter the number represents an amino acid residue before thesubstitution):

(k) F241A, (l) D265A, and (m) V264A

defined according to the EU numbering can also be appropriately used.

An antigen-binding molecule having an Fc region derived from an IgG4antibody Fc region by any of the following substitution groups of theconstituent amino acids (the number represents the position of an aminoacid residue defined according to the EU numbering; the one-letter aminoacid code positioned before the number represents an amino acid residuebefore the substitution; and the one-letter amino acid code positionedafter the number represents an amino acid residue before thesubstitution):

(n) L235A, G237A, and E318A, (o) L235E, and (p) F234A and L235A

defined according to the EU numbering can also be appropriately used.

Other preferred examples thereof include antigen-binding moleculeshaving an Fc region derived from the Fc region of a naturally occurringhuman IgG1 antibody by the substitution of any of the followingconstituent amino acids: amino acids at positions 233, 234, 235, 236,237, 327, 330, and 331 defined according to the EU numbering, by anamino acid at the corresponding EU numbering position in the Fc regionof the counterpart IgG2 or IgG4.

Other preferred examples thereof include antigen-binding moleculeshaving an Fc region derived from the Fc region of a naturally occurringhuman IgG1 antibody by the substitution of any one or more of thefollowing constituent amino acids: amino acids at positions 234, 235,and 297 defined according to the EU numbering, by a different aminoacid. The type of the amino acid present after the substitution is notparticularly limited. An antigen-binding molecule having an Fc regionwith any one or more of amino acids at positions 234, 235, and 297substituted by alanine is particularly preferred.

Other preferred examples thereof include antigen-binding moleculeshaving an Fc region derived from an IgG1 antibody Fc region by thesubstitution of the constituent amino acid at position 265 definedaccording to the EU numbering, by a different amino acid. The type ofthe amino acid present after the substitution is not particularlylimited. An antigen-binding molecule having an Fc region with an aminoacid at position 265 substituted by alanine is particularly preferred.

One preferred form of the “antigen-binding molecule” of the presentinvention can be, for example, a multispecific antibody comprising theantibody variable region of the present invention.

A technique of suppressing the unintended association between H chainsby introducing electric charge repulsion to the interface between thesecond constant domains (CH2) or the third constant domains (CH3) of theantibody H chains (WO2006/106905) can be applied to association for themultispecific antibody.

In the technique of suppressing the unintended association between Hchains by introducing electric charge repulsion to the CH2 or CH3interface, examples of amino acid residues contacting with each other atthe interface between the H chain constant domains can include a residueat EU numbering position 356, a residue at EU numbering position 439, aresidue at EU numbering position 357, a residue at EU numbering position370, a residue at EU numbering position 399, and a residue at EUnumbering position 409 in one CH3 domain, and their partner residues inanother CH3 domain.

More specifically, for example, an antibody comprising two H chain CH3domains can be prepared as an antibody in which one to three pairs ofamino acid residues selected from the following amino acid residue pairs(1) to (3) in the first H chain CH3 domain carry the same electriccharge: (1) amino acid residues at EU numbering positions 356 and 439contained in the H chain CH3 domain; (2) amino acid residues at EUnumbering positions 357 and 370 contained in the H chain CH3 domain; and(3) amino acid residues at EU numbering positions 399 and 409 containedin the H chain CH3 domain.

The antibody can be further prepared as an antibody in which one tothree pairs of amino acid residues are selected from the amino acidresidue pairs (1) to (3) in the second H chain CH3 domain different fromthe first H chain CH3 domain so as to correspond to the amino acidresidue pairs (1) to (3) carrying the same electric charge in the firstH chain CH3 domain and to carry opposite electric charge from theircorresponding amino acid residues in the first H chain CH3 domain.

Each amino acid residue described in the pairs (1) to (3) is locatedclose to its partner in the associated H chains. Those skilled in theart can find positions corresponding to the amino acid residuesdescribed in each of the pairs (1) to (3) as to the desired H chain CH3domains or H chain constant domains by homology modeling or the likeusing commercially available software and can appropriately alter aminoacid residues at the positions.

In the antibody described above, each of the “amino acid residuescarrying electric charge” is preferably selected from, for example,amino acid residues included in any of the following groups (a) and (b):

(a) glutamic acid (E) and aspartic acid (D); and(b) lysine (K), arginine (R), and histidine (H).

In the antibody described above, the phrase “carrying the same electriccharge” means that, for example, all of two or more amino acid residuesare amino acid residues included in any one of the groups (a) and (b).The phrase “carrying opposite electric charge” means that, for example,at least one amino acid residue among two or more amino acid residuesmay be an amino acid residue included in any one of the groups (a) and(b), while the remaining amino acid residue(s) is amino acid residue(s)included in the other group.

In a preferred embodiment, the antibody may have the first H chain CH3domain and the second H chain CH3 domain cross-linked through adisulfide bond.

The amino acid residue to be altered according to the present inventionis not limited to the amino acid residues in the antibody variableregion or the antibody constant region mentioned above. Those skilled inthe art can find amino acid residues constituting the interface as to apolypeptide variant or a heteromultimer by homology modeling or the likeusing commercially available software and can alter amino acid residuesat the positions so as to regulate the association.

The association for the multispecific antibody of the present inventioncan also be carried out by an alternative technique known in the art. Anamino acid side chain present in the variable domain of one antibody Hchain is substituted by a larger side chain (knob), and its partneramino acid side chain present in the variable domain of the other Hchain is substituted by a smaller side chain (hole). The knob can beplaced into the hole to efficiently associate the polypeptides of the Fcdomains differing in amino acid sequence (WO1996/027011; Ridgway J B etal., Protein Engineering (1996) 9, 617-621; and Merchant A M et al.Nature Biotechnology (1998) 16, 677-681).

In addition to this technique, a further alternative technique known inthe art may be used for forming the multispecific antibody of thepresent invention. A portion of CH3 of one antibody H chain is convertedto its counterpart IgA-derived sequence, and its complementary portionin CH3 of the other H chain is converted to its counterpart IgA-derivedsequence. Use of the resulting strand-exchange engineered domain CH3 cancause efficient association between the polypeptides differing insequence through complementary CH3 association (Protein EngineeringDesign & Selection, 23; 195-202, 2010). By use of this technique knownin the art, the multispecific antibody of interest can also beefficiently formed.

Alternatively, the multispecific antibody may be formed by, for example,an antibody preparation technique using antibody CH1-CL association andVH-VL association as described in WO2011/028952, a technique ofpreparing a bispecific antibody using separately prepared monoclonalantibodies (Fab arm exchange) as described in WO2008/119353 andWO2011/131746, a technique of controlling the association betweenantibody heavy chain CH3 domains as described in WO2012/058768 andWO2013/063702, a technique of preparing a bispecific antibodyconstituted by two types of light chains and one type of heavy chain asdescribed in WO2012/023053, or a technique of preparing a bispecificantibody using two bacterial cell lines each expressing an antibodyhalf-molecule consisting of one H chain and one L chain as described inChristoph et al. (Nature Biotechnology Vol. 31, p. 753-758 (2013)). Inaddition to these association techniques, CrossMab technology, a knownhetero light chain association technique of associating a light chainforming a variable region binding to a first epitope and a light chainforming a variable region binding to a second epitope to a heavy chainforming the variable region binding to the first epitope and a heavychain forming the variable region binding to the second epitope,respectively (Scaefer et al., Proc. Natl. Acad. Sci. U.S.A. (2011) 108,11187-11192), can also be used for preparing a multispecific ormultiparatopic antigen-binding molecule provided by the presentinvention. Examples of the technique of preparing a bispecific antibodyusing separately prepared monoclonal antibodies can include a methodwhich involves promoting antibody heterodimerization by placingmonoclonal antibodies with a particular amino acid substituted in aheavy chain CH3 domain under reductive conditions to obtain the desiredbispecific antibody. Examples of the amino acid substitution sitepreferred for this method can include a residue at EU numbering position392 and a residue at EU numbering position 397 in the CH3 domain.Furthermore, the bispecific antibody can also be prepared by use of anantibody in which one to three pairs of amino acid residues selectedfrom the following amino acid residue pairs (1) to (3) in the first Hchain CH3 domain carry the same electric charge: (1) amino acid residuesat EU numbering positions 356 and 439 contained in the H chain CH3domain; (2) amino acid residues at EU numbering positions 357 and 370contained in the H chain CH3 domain; and (3) amino acid residues at EUnumbering positions 399 and 409 contained in the H chain CH3 domain. Thebispecific antibody can also be prepared by use of the antibody in whichone to three pairs of amino acid residues are selected from the aminoacid residue pairs (1) to (3) in the second H chain CH3 domain differentfrom the first H chain CH3 domain so as to correspond to the amino acidresidue pairs (1) to (3) carrying the same electric charge in the firstH chain CH3 domain and to carry opposite electric charge from theircorresponding amino acid residues in the first H chain CH3 domain.

Even if the multispecific antibody of interest cannot be formedefficiently, the multispecific antibody of the present invention may beobtained by the separation and purification of the multispecificantibody of interest from among produced antibodies. For example, thepreviously reported method involves introducing amino acid substitutionto the variable domains of two types of H chains to impart theretodifference in isoelectric point so that two types of homodimers and theheterodimerized antibody of interest can be separately purified byion-exchanged chromatography (WO2007114325). A method using protein A topurify a heterodimerized antibody consisting of a mouse IgG2a H chaincapable of binding to protein A and a rat IgG2b H chain incapable ofbinding to protein A has previously been reported as a method forpurifying the heterodimer (WO98050431 and WO95033844). Alternatively,amino acid residues at EU numbering positions 435 and 436 thatconstitute the protein A-binding site of IgG may be substituted by aminoacids, such as Tyr and His, which offer the different strength ofprotein A binding, and the resulting H chain is used to change theinteraction of each H chain with protein A. As a result, only theheterodimerized antibody can be efficiently purified by use of a proteinA column.

A plurality of, for example, two or more of these techniques may be usedin combination. Also, these techniques can be appropriately appliedseparately to the two H chains to be associated. On the basis of, butseparately from the form thus altered, the antigen-binding molecule ofthe present invention may be prepared as an antigen-binding moleculehaving an amino acid sequence identical thereto.

The alteration of an amino acid sequence can be performed by variousmethods known in the art. Examples of these methods that may beperformed can include, but are not limited to, methods such assite-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y,and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual ambermethod for site-directed mutagenesis. Gene 152, 271-275; Zoller, M J,and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNAfragments cloned into M13 vectors. Methods Enzymol. 100, 468-500;Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz,H J (1984) The gapped duplex DNA approach to oligonucleotide-directedmutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, andFritz H J (1987) Oligonucleotide-directed construction of mutations viagapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, T A (1985)Rapid and efficient site-specific mutagenesis without phenotypicselection. Proc Natl Acad Sci USA. 82, 488-492), PCR mutagenesis, andcassette mutagenesis.

The “antigen-binding molecule” of the present invention may be anantibody fragment that comprises both of a heavy chain and a light chainconstituting the “antibody variable region” of the present invention ina single polypeptide chain, but lacks a constant region. Such anantibody fragment may be, for example, diabody (Db), a single-chainantibody, or sc(Fab′)2.

Db is a dimer constituted by two polypeptide chains (e.g., Holliger P etal., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; andWO93/11161). These polypeptide chains are linked through a linker asshort as, for example, approximately 5 residues, such that an L chainvariable domain (VL) and an H chain variable domain (VH) on the samepolypeptide chain cannot be paired with each other.

Because of this short linker, VL and VH encoded on the same polypeptidechain cannot form single-chain Fv and instead, are dimerized with VH andVL, respectively, on another polypeptide chain, to form twoantigen-binding sites.

Examples of the single-chain antibody include sc(Fv)2. The sc(Fv)2 is asingle-chain antibody having one chain constituted by four variabledomains, i.e., two VLs and two VHs, linked via linkers such as peptidelinkers (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHsand VLs may be derived from different monoclonal antibodies. Preferredexamples thereof include bispecific sc(Fv)2, which recognizes two typesof epitopes present in the same antigen, as disclosed in Journal ofImmunology (1994) 152 (11), 5368-5374. The sc(Fv)2 can be prepared by amethod generally known to those skilled in the art. For example, thesc(Fv)2 can be prepared by connecting two scFvs via a linker such as apeptide linker.

Examples of the configuration of the antigen-binding domainsconstituting the sc(Fv)2 described herein include an antibody in whichtwo VHs and two VLs are aligned as VH, VL, VH, and VL (i.e.,[VH]-linker-[VL]-linker-[VH]-linker-[VL]) in this order starting at theN-terminus of the single-chain polypeptide. The order of two VHs and twoVLs is not particularly limited to the configuration described above andmay be any order of arrangement. Examples thereof can also include thefollowing arrangements:

[VL]-linker-[VH]-linker-[VH]-linker-[VL],[VH]-linker-[VL]-linker-[VL]-linker-[VH],[VH]-linker-[VH]-linker-[VL]-linker-[VL],[VL]-linker-[VL]-linker-[VH]-linker-[VH], and[VL]-linker-[VH]-linker-[VL]-linker-[VH].

The molecular form of the sc(Fv)2 is also described in detail inWO2006/132352. On the basis of the description therein, those skilled inthe art can appropriately prepare the desired sc(Fv)2 in order toprepare the antigen-binding molecule disclosed in the presentspecification.

The antigen-binding molecule of the present invention may be conjugatedwith a carrier polymer such as PEG or an organic compound such as ananticancer agent. Also, a sugar chain can be preferably added to theantigen-binding molecule of the present invention by the insertion of aglycosylation sequence for the purpose of producing the desired effects.

For example, an arbitrary peptide linker that can be introduced bygenetic engineering, or a synthetic compound linker (e.g., a linkerdisclosed in Protein Engineering, 9 (3), 299-305, 1996) can be used asthe linker to link the antibody variable domains. In the presentinvention, a peptide linker is preferred. The length of the peptidelinker is not particularly limited and can be appropriately selected bythose skilled in the art according to the purpose. The length ispreferably 5 or more amino acids (the upper limit is not particularlylimited and is usually 30 or less amino acids, preferably 20 or lessamino acids), particularly preferably 15 amino acids. When the sc(Fv)2contains three peptide linkers, all of these peptide linkers used mayhave the same lengths or may have different lengths.

Examples of the peptide linker can include

Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, (SEQ ID NO: 5) Gly-Gly-Gly-Ser,(SEQ ID NO: 6) Ser-Gly-Gly-Gly, (SEQ ID NO: 7) Gly-Gly-Gly-Gly-Ser,(SEQ ID NO: 8) Ser-Gly-Gly-Gly-Gly, (SEQ ID NO: 9)Gly-Gly-Gly-Gly-Gly-Ser, (SEQ ID NO: 10) Ser-Gly-Gly-Gly-Gly-Gly,(SEQ ID NO: 11) Gly-Gly-Gly-Gly-Gly-Gly-Ser, (SEQ ID NO: 12)Ser-Gly-Gly-Gly-Gly-Gly-Gly, (Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 7))n, and(Ser-Gly-Gly-Gly-Gly(SEQ ID NO: 8))n,wherein n is an integer of 1 or larger.However, the length or sequence of the peptide linker can beappropriately selected by those skilled in the art according to thepurpose.

The synthetic compound linker (chemical cross-linking agent) is across-linking agent usually used in the cross-linking of peptides, forexample, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate(DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), orbis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

These cross-linking agents are commercially available.

Three linkers are usually necessary for linking four antibody variabledomains. All of these linkers used may be the same linkers or may bedifferent linkers.

The F(ab′)2 comprises two light chains and two heavy chains containing aconstant region (CH1 domains and a portion of CH2 domains) so as to formthe interchain disulfide bond between these two heavy chains. TheF(ab′)2 constituting a polypeptide associate disclosed in the presentspecification can be preferably obtained by the partial digestion of,for example, a whole monoclonal antibody having the desiredantigen-binding domains with a proteolytic enzyme such as pepsinfollowed by the removal of an Fc fragment adsorbed on a protein Acolumn. Such a proteolytic enzyme is not particularly limited as long asthe enzyme is capable of digesting a whole antibody to restrictivelyform F(ab′)₂ under appropriately set reaction conditions (e.g., pH) ofthe enzyme. Examples thereof can include pepsin and ficin.

The antigen-binding molecule of the present invention can furthercontain additional alteration in addition to the amino acid alterationmentioned above. The additional alteration can be selected from, forexample, amino acid substitution, deletion, and modification, and acombination thereof.

For example, the antigen-binding molecule of the present invention canbe further altered arbitrarily, substantially without changing theintended functions of the molecule. Such a mutation can be performed,for example, by the conservative substitution of amino acid residues.Alternatively, even alteration to change the intended functions of theantigen-binding molecule of the present invention may be carried out aslong as the functions changed by such alteration fall within the objectof the present invention.

The alteration of an amino acid sequence according to the presentinvention also includes posttranslational modification. Specifically,the posttranslational modification can refer to the addition or deletionof a sugar chain. The antigen-binding molecule of the present invention,for example, having an IgG1-type constant region, can have a sugarchain-modified amino acid residue at EU numbering position 297. Thesugar chain structure for use in the modification is not limited. Ingeneral, antibodies expressed by eukaryotic cells involve sugar chainmodification in their constant regions. Thus, antibodies expressed bythe following cells are usually modified with some sugar chain:

mammalian antibody-producing cells; andeukaryotic cells transformed with expression vectors comprisingantibody-encoding DNAs.

In this context, the eukaryotic cells include yeast and animal cells.For example, CHO cells or HEK293H cells are typical animal cells fortransformation with expression vectors comprising antibody-encodingDNAs. On the other hand, the antibody of the present invention alsoincludes antibodies lacking sugar chain modification at the position.The antibodies having sugar chain-unmodified constant regions can beobtained by the expression of genes encoding these antibodies inprokaryotic cells such as E. coli.

The additional alteration according to the present invention may be morespecifically, for example, the addition of sialic acid to a sugar chainin an Fc region (mAbs. 2010 September-October; 2 (5): 519-27).

When the antigen-binding molecule of the present invention has an Fcregion, for example, amino acid substitution to improve binding activityagainst FcRn (J Immunol. 2006 Jan. 1; 176 (1): 346-56; J Biol Chem. 2006Aug. 18; 281 (33): 23514-24; Int Immunol. 2006 December; 18 (12):1759-69; Nat Biotechnol. 2010 February; 28 (2): 157-9; WO2006/019447;WO2006/053301; and WO2009/086320) or amino acid substitution to improveantibody heterogeneity or stability ((WO2009/041613)) may be addedthereto.

In the present invention, the term “antibody” is used in the broadestsense and also includes any antibody such as monoclonal antibodies(including whole monoclonal antibodies), polyclonal antibodies, antibodyvariants, antibody fragments, multispecific antibodies (e.g., bispecificantibodies), chimeric antibodies, and humanized antibodies as long asthe antibody exhibits the desired biological activity.

The antibody of the present invention is not limited by the type of itsantigen, its origin, etc., and may be any antibody. Examples of theorigin of the antibody can include, but are not particularly limited to,human antibodies, mouse antibodies, rat antibodies, and rabbitantibodies.

The antibody can be prepared by a method well known to those skilled inthe art. For example, the monoclonal antibodies may be produced by ahybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) or arecombination method (U.S. Pat. No. 4,816,567). Alternatively, themonoclonal antibodies may be isolated from phage-displayed antibodylibraries (Clackson et al., Nature 352: 624-628 (1991); and Marks etal., J. Mol. Biol. 222: 581-597 (1991)). Also, the monoclonal antibodiesmay be isolated from single B cell clones (N. Biotechnol. 28 (5):253-457 (2011)).

The humanized antibodies are also called reshaped human antibodies.Specifically, for example, a humanized antibody consisting of anon-human animal (e.g., mouse) antibody CDR-grafted human antibody isknown in the art. General gene recombination approaches are also knownfor obtaining the humanized antibodies. Specifically, for example,overlap extension PCR is known in the art as a method for grafting mouseantibody CDRs to human FRs.

DNAs encoding antibody variable domains each comprising three CDRs andfour FRs linked and DNAs encoding human antibody constant domains can beinserted into expression vectors such that the variable domain DNAs arefused in frame with the constant domain DNAs to prepare vectors forhumanized antibody expression. These vectors having the inserts aretransferred to hosts to establish recombinant cells. Then, therecombinant cells are cultured for the expression of the DNAs encodingthe humanized antibodies to produce the humanized antibodies into thecultures of the cultured cells (see European Patent Publication No. EP239400 and International Publication No. WO1996/002576).

If necessary, FR amino acid residue(s) may be substituted such that theCDRs of the reshaped human antibody form an appropriate antigen-bindingsite. For example, the amino acid sequence of FR can be mutated by theapplication of the PCR method used in the mouse CDR grafting to thehuman FRs.

The desired human antibody can be obtained by DNA immunization usingtransgenic animals having all repertoires of human antibody genes (seeInternational Publication Nos. WO1993/012227, WO1992/003918,WO1994/002602, WO1994/025585, WO1996/034096, and WO1996/033735) asimmunized animals.

In addition, a technique of obtaining human antibodies by panning usinghuman antibody libraries is also known. For example, a human antibody Vregion is expressed as a single-chain antibody (scFv) on the surface ofphages by a phage display method. A phage expressing antigen-bindingscFv can be selected. The gene of the selected phage can be analyzed todetermine a DNA sequence encoding the V region of the antigen-bindinghuman antibody. After the determination of the DNA sequence of theantigen-binding scFv, the V region sequence can be fused in frame withthe sequence of the desired human antibody C region and then inserted toappropriate expression vectors to prepare expression vectors. Theexpression vectors are transferred to the preferred expression cellslisted above for the expression of the genes encoding the humanantibodies to obtain the human antibodies. These methods are alreadyknown in the art (see International Publication Nos. WO1992/001047,WO1992/020791, WO1993/006213, WO1993/011236, WO1993/019172,WO1995/001438, and WO1995/015388).

The variable regions constituting the antibody of the present inventioncan be variable regions that recognize an arbitrary antigen.

In the present specification, the “antigen” is not particularly limitedand may be any antigen. Examples of the antigen include 17-IA, 4-1 BB,4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33,ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C,Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RITA, ActivinRIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF,AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin,alpha1-antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, aminin,amylin, amyloid beta, amyloid immunoglobulin heavy chain variableregion. amyloid immunoglobulin light chain variable region, Androgen,ANG, angiotensinogen, Angiopoietin ligand-2, anti-Id, antithrombinIII,Anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP,APRIL, AR, ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor,Atrial natriuretic peptide, atrial natriuretic peptides A, atrialnatriuretic peptides B, atrial natriuretic peptides C, av/b3 integrin,Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracia protectiveantigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA,BDNF, b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, B-lymphocyte Stimulator (BIyS), BMP, BMP-2 (BMP-2a),BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1),BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3),BMPs, BOK, Bombesin, Bone-derived neurotrophic factor, bovine growthhormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesionmolecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a),CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX,carcinoembryonic antigen (CEA), carcinoma-associated antigen,Cardiotrophin-1, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, CathepsinD, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1/I-309,CCL11/Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2,CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1,CCL20/MIP-3-alpha, CCL21/SLC, CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2,CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-alpha,CCL3L1/LD-78-beta, CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10, CCL7/MCP-3,CCL8/MCP-2, CCL9/10/MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4,CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c,CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15,CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26,CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34,CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5,CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74,CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP,CGRP receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinumtoxin, Clostridium difficile toxin, Clostridium perfringens toxin,c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complementfactor D, corticosteroid-binding globulin, Colony stimulating factor-1receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4,CX3CL1/Fractalkine, CX3CR1, CXCL, CXCL1/Gro-alpha, CXCL10, CXCL11/I-TAC,CXCL12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine.CXCL16, CXCL16, CXCL2/Gro-beta CXCL3/Gro-gamma, CXCL3, CXCL4/PF4,CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig,CXCL1O/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatinC, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brainIGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidaseIV, DK1, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2,EDAR, EGF, EGFR (ErbB-1), EGF like domain containing protein 7,Elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, endothelinreceptor, endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2,eotaxini, EpCAM, Ephrin B2/EphB4, Epha2 tyrosine kinase receptor,epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosinekinase receptor, ERCC, EREG, erythropoietin (EPO), Erythropoietinreceptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12,F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, factor VIII,Factor VIIIc, Fas, FcalphaR, FcepsilonRI, Fcgammallb, FcgammaRI,FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, Ferritin, FGF,FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic,FGFR, FGFR-3, Fibrin, fibroblast activation protein (FAP), fibroblastgrowth factor, fibroblast growth factor-10, fibronectin, FL, FLIP,Flt-3, FLT3 ligand, Folate receptor, follicle stimulating hormone (FSH),Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2,GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3(Vgr-2), GDF-5 (BMP-14/CDMP-1), GDF-6 (BMP-13/CDMP-2), GDF-7(BMP-12/CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF,GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-β1, gH envelope glycoprotein,GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor,Glut 4, Glutamate carboxypeptidase II, glycoprotein hormone receptors,glycoprotein IIb/IIIa (GP IIb/IIIa), Glypican-3, GM-CSF, GM-CSFreceptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, Growthhormone releasing factor, GRO-β, GRO-γ, H. pylori, Hapten (NP-cap orNIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, heparincofactor II, hepatic growth factor, Bacillus anthracis protectiveantigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1,Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus(HSV) gB glycoprotein, HGF, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV envelope proteins such asGP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1,HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, humancytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin,human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP,ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta, IFN-gamma, IgA,IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2,IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptors, IL-11, IL-11 receptors,IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors,IL-16, IL-16 receptors, IL-17, IL-17 receptors, IL-18 (IGIF), IL-18receptors, IL-lalpha, IL-1beta, IL-1 receptors, IL-2, IL-2 receptors,IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors,IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3receptors, IL-4, IL-4 receptors IL-5, IL-5 receptors, IL-6, IL-6receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha,INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gammareceptors, IFN type-I, IFN type-I receptor, influenza, inhibin, Inhibinα, Inhibin β, iNOS, insulin, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, insulin-like growth factor 2, insulin-likegrowth factor binding proteins, integrin, integrin alpha2, integrinalpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha-V/beta-3,integrin alpha-V/beta-6, integrin alpha4/beta7, integrin alpha5/beta1,integrin alpha5/beta3, integrin alpha5/beta6, integrin alpha σ (alphaV),integrin alpha θ, integrin beta1, integrin beta2, integrinbeta3(GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11,Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5,Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, KallikreinL4, kallistatin, KC, KDR, Keratinocyte Growth Factor (KGF), KeratinocyteGrowth Factor-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kitligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin,islet-amyloid polypeptide), LAP (TGF-1), latency associated peptide,Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2,Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Yrelated antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT,lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lungsurfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin BetaReceptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF),MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3,MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC (69 a.a.), megsin,Mer, MET tyrosine kinase receptor family, METALLOPROTEASES, Membraneglycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC (HLA-DR),microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4,MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein,monocyte colony inhibitory factor, mouse gonadotropin-associatedpeptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud),Muellerian-inhibiting substance, Mug, MuSK, Myelin associatedglycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP,Nanobody, NAP, NAP-2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin, Neuralcell adhesion molecule, neroserpin, Neuronal growth factor (NGF),Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin 1, Neurturin,NGF-beta, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO,Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from thehepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1,Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductivefactors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr,parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA,PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1,PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, Placentagrowth factor, placental alkaline phosphatase (PLAP), placentallactogen, plasminogen activator inhibitor-1, platelet-growth factor,plgR, PLP, poly glycol chains of different size (e.g. PEG-20, PEG-30,PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmedcell death protein 1, proinsulin, prolactin, Proprotein convertase PC9,prorelaxin, prostate specific membrane antigen (PSMA), Protein A,Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN,PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK,RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoidfactors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, 5100, RON-8, SCF/KL, SCGF,Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin,sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF,SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcallipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF),streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72(tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptoralpha/beta, TdT, TECK, TEM1, TEMS, TEM7, TEM8, Tenascin, TERT,testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-betaRIII, TGF-beta R1 (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4,TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromallymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH),thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor,tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo,TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta,TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2/DR4),TNFRSF10B (TRAIL R2 DR5/KILLER/TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3DcR1/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODFR/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAK R FN14),TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEMATAR/HveA/LIGHT R/TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA),TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT),TNFRSF1A (TNF R1 CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80),TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII/TNFC R), TNFRSF4 (OX40 ACT35/TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6(Fas Apo-1/APT1/CD95), TNFRSF6B (DcR3 M68/TR6), TNFRSF7 (CD27), TNFRSF8(CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL R1 TNFRH1),TNFSF10 (TRAIL Apo-2 Ligand/TL2), TNFSF11 (TRANCE/RANK Ligand ODF/OPGLigand), TNFSF12 (TWEAK Apo-3 Ligand/DR3 Ligand), TNFSF13 (APRIL TALL2),TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEMLigand/LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand/TL6),TNFSF1A (TNF-a Conectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3(LTb TNFC/p33), TNFSF4 (OX40 Ligand gp34/TXGP1), TNFSF5 (CD40 LigandCD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand/APT1Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9(4-1 BB Ligand CD137 Ligand), TNF-α, TNF-β, TNIL-I, toxic metabolite,TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrinreceptor, transforming growth factors (TGF) such as TGF-alpha andTGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk,TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor(TNF), tumor-associated antigen CA 125, tumor-associated antigenexpressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1,Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin,VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1),VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viralantigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNRintegrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B,WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6,WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1-beta,XCL1/Lymphotactin, XCR1, XEDAR, XIAP, and XPD.

One of the two variable regions of the antibody included in theantigen-binding molecule of the present invention is capable of bindingto two different antigens, but cannot bind to these antigens at the sametime. The “first antigen” or the “second antigen” to which this variableregion binds is preferably, for example, an immunocyte surface molecule(e.g., a T cell surface molecule, an NK cell surface molecule, adendritic cell surface molecule, a B cell surface molecule, an NKT cellsurface molecule, an MDSC cell surface molecule, and a macrophagesurface molecule), or an antigen expressed not only on tumor cells,tumor vessels, stromal cells, and the like but on normal tissues(integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokinereceptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF,etc.). As for the combination of the “first antigen” and the “secondantigen”, preferably, any one of the first antigen and the secondantigen is, for example, a molecule specifically expressed on a T cell,and the other antigen is a molecule expressed on the surface of a T cellor any other immunocyte. In another embodiment of the combination of the“first antigen” and the “second antigen”, preferably, any one of thefirst antigen and the second antigen is, for example, a moleculespecifically expressed on a T cell, and the other antigen is a moleculethat is expressed on an immunocyte and is different from thepreliminarily selected antigen. Specific examples of the moleculespecifically expressed on a T cell include CD3 and T cell receptors.Particularly, CD3 is preferred. In the case of, for example, human CD3,a site in the CD3 to which the antigen-binding molecule of the presentinvention binds may be any epitope present in a γ chain, δ chain, or εchain sequence constituting the human CD3. Particularly, an epitopepresent in the extracellular region of an c chain in a human CD3 complexis preferred. The polynucleotide sequences of the γ chain, δ chain, andc chain structures constituting CD3 are shown in SEQ ID NOs: 83(NM_000073.2), 85 (NM_000732.4), and 87 (NM_000733.3), and thepolypeptide sequences thereof are shown in SEQ ID NOs: 84 (NP_000064.1),86 (NP_000723.1), and 88 (NP_000724.1) (RefSeq registration numbers areshown within the parentheses). Examples of the other antigen include Fcγreceptors, TLR, lectin, IgA, immune checkpoint molecules, TNFsuperfamily molecules, TNFR superfamily molecules, and NK receptormolecules.

Of the two variable regions of the antibody included in theantigen-binding molecule of the present invention, the other variableregion binds to a “third antigen” that is different from the “firstantigen” and the “second antigen” mentioned above. The third antigen ispreferably, for example, a tumor cell-specific antigen and also includesan antigen expressed in association with the malignant alteration ofcells as well as an abnormal sugar chain that appears on cell surface ora protein molecule during the malignant transformation of cells.Specific examples thereof include ALK receptor (pleiotrophin receptor),pleiotrophin, KS 1/4 pancreatic cancer antigen, ovary cancer antigen(CA125), prostatic acid phosphate, prostate-specific antigen (PSA),melanoma-associated antigen p97, melanoma antigen gp75,high-molecular-weight melanoma antigen (HMW-MAA), prostate-specificmembrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelialmucin antigen, human milk fat globule antigen, colorectaltumor-associated antigen (e.g., CEA, TAG-72, C017-1A, GICA 19-9, CTA-1,and LEA), Burkitt's lymphoma antigen 38.13, CD19, human B lymphomaantigen CD20, CD33, melanoma-specific antigen (e.g., ganglioside GD2,ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specifictransplantation antigen (TSTA), T antigen, virus-induced tumor antigen(e.g., envelope antigens of DNA tumor virus and RNA tumor virus), colonCEA, oncofetal antigen α-fetoprotein (e.g., oncofetal trophoblasticglycoprotein 5T4 and oncofetal bladder tumor antigen), differentiationantigen (e.g., human lung cancer antigens L6 and L20), fibrosarcomaantigen, human T cell leukemia-associated antigen Gp37, newbornglycoprotein, sphingolipid, breast cancer antigen (e.g., EGFR(epithelial growth factor receptor)), NY-BR-16, NY-BR-16 and HER2antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant humanlymphocyte antigen APO-1, differentiation antigen such as I antigenfound in fetal erythrocytes, primary endoderm I antigen found in adulterythrocytes, I (Ma) found in embryos before transplantation or gastriccancer, M18 found in mammary gland epithelium, M39, SSEA-1 found in bonemarrow cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectalcancer, TRA-1-85 (blood group H), SCP-1 found in testis and ovarycancers, C14 found in colon cancer, F3 found in lung cancer, AH6 foundin gastric cancer, Y hapten, Ley found in embryonic cancer cells, TL5(blood group A), EGF receptor found in A431 cells, E1 series (bloodgroup B) found in pancreatic cancer, FC10.2 found in embryonic cancercells, gastric cancer antigen, CO-514 (blood group Lea) found inadenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood group Leb),G49 found in A431 cell EGF receptor, MH2 (blood group ALeb/Ley) found incolon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7found in bone marrow cells, R24 found in melanoma, 4.2, GD3, D1.1,OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 found in embryonic cancer cells,SSEA-3 and SSEA-4 found in 4-cell to 8-cell embryos, cutaneous T celllymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen,colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A,adenocarcinoma antigen ART1, paraneoplastic associatedbrain-testis-cancer antigen (onconeuronal antigen MA2 and paraneoplasticneuronal antigen), neuro-oncological ventral antigen 2 (NOVA2), bloodcell cancer antigen gene 520, tumor-associated antigen CO-029,tumor-associated antigen MAGE-C1 (cancer/testis antigen CT7), MAGE-B1(MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b MAGE-X2,cancer-testis antigen (NY-EOS-1), YKL-40, and any fragment of thesepolypeptides, and modified structures thereof (aforementioned modifiedphosphate groups, sugar chains, etc.), EpCAM, EREG, CA19-9, CA15-3,sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

The antigen-binding molecule of the present invention can be produced bya method generally known to those skilled in the art. For example, theantibody can be prepared by a method given below, though the method forpreparing the antibody of the present invention is not limited thereto.Many combinations of host cells and expression vectors are known in theart for antibody preparation by the transfer of isolated genes encodingpolypeptides into appropriate hosts. All of these expression systems canbe applied to the isolation of the antigen-binding molecule of thepresent invention. In the case of using eukaryotic cells as the hostcells, animal cells, plant cells, or fungus cells can be appropriatelyused. Specifically, examples of the animal cells can include thefollowing cells:

(1) mammalian cells such as CHO (Chinese hamster ovary cell line), COS(monkey kidney cell line), myeloma cells (Sp2/O, NSO, etc.), BHK (babyhamster kidney cell line), HEK293 (human embryonic kidney cell line withsheared adenovirus (Ad)5 DNA), PER. C6 cell (human embryonic retinalcell line transformed with the adenovirus type 5 (Ad5) E1A and E1Bgenes), Hela, and Vero (Current Protocols in Protein Science (May, 2001,Unit 5.9, Table 5.9.1));(2) amphibian cells such as Xenopus oocytes; and(3) insect cells such as sf9, sf21, and Tn5.

The antibody can also be prepared using E. coli (mAbs 2012 March-April;4 (2): 217-225) or yeast (WO2000023579). The antibody prepared using E.coli is not glycosylated. On the other hand, the antibody prepared usingyeast is glycosylated.

An antibody heavy chain-encoding DNA that encodes a heavy chain with oneor more amino acid residues in a variable domain substituted bydifferent amino acids of interest, and a DNA encoding a light chain ofthe antibody are expressed. The DNA that encodes a heavy chain or alight chain with one or more amino acid residues in a variable domainsubstituted by different amino acids of interest can be obtained, forexample, by obtaining a DNA encoding an antibody variable domainprepared by a method known in the art against a certain antigen, andappropriately introducing substitution such that codons encoding theparticular amino acids in the domain encode the different amino acids ofinterest.

Alternatively, a DNA encoding a protein in which one or more amino acidresidues in an antibody variable domain prepared by a method known inthe art against a certain antigen are substituted by different aminoacids of interest may be designed in advance and chemically synthesizedto obtain the DNA that encodes a heavy chain with one or more amino acidresidues in a variable domain substituted by different amino acids ofinterest. The amino acid substitution site and the type of thesubstitution are not particularly limited. Examples of the regionpreferred for the amino acid alteration include solvent-exposed regionsand loops in the variable region. Among others, CDR1, CDR2, CDR3, FR3,and loops are preferred. Specifically, Kabat numbering positions 31 to35, 50 to 65, 71 to 74, and 95 to 102 in the H chain variable domain andKabat numbering positions 24 to 34, 50 to 56, and 89 to 97 in the Lchain variable domain are preferred. Kabat numbering positions 31, 52ato 61, 71 to 74, and 97 to 101 in the H chain variable domain and Kabatnumbering positions 24 to 34, 51 to 56, and 89 to 96 in the L chainvariable domain are more preferred.

The amino acid alteration is not limited to the substitution and may bedeletion, addition, insertion, or modification, or a combinationthereof.

The DNA that encodes a heavy chain with one or more amino acid residuesin a variable domain substituted by different amino acids of interestcan also be produced as separate partial DNAs. Examples of thecombination of the partial DNAs include, but are not limited to: a DNAencoding a variable domain and a DNA encoding a constant domain; and aDNA encoding a Fab domain and a DNA encoding an Fc domain. Likewise, thelight chain-encoding DNA can also be produced as separate partial DNAs.

These DNAs can be expressed by the following method: for example, a DNAencoding a heavy chain variable domain, together with a DNA encoding aheavy chain constant domain, is integrated to an expression vector toconstruct a heavy chain expression vector. Likewise, a DNA encoding alight chain variable domain, together with a DNA encoding a light chainconstant domain, is integrated to an expression vector to construct alight chain expression vector. These heavy chain and light chain genesmay be integrated to a single vector.

The DNA encoding the antibody of interest is integrated to expressionvectors so as to be expressed under the control of expression controlregions, for example, an enhancer and a promoter. Next, host cells aretransformed with the resulting expression vectors and allowed to expressantibodies. In this case, appropriate hosts and expression vectors canbe used in combination.

Examples of the vectors include M13 series vectors, pUC series vectors,pBR322, pBluescript, and pCR-Script. In addition to these vectors, forexample, pGEM-T, pDIRECT, or pT7 can also be used for the purpose ofcDNA subcloning and excision.

Particularly, expression vectors are useful for using the vectors forthe purpose of producing the antibody of the present invention. Forexample, when the host is E. coli such as JM109, DH5a, HB101, orXL1-Blue, the expression vectors indispensably have a promoter thatpermits efficient expression in E. coli, for example, lacZ promoter(Ward et al., Nature (1989) 341, 544-546; and FASEB J. (1992) 6,2422-2427, which are incorporated herein by reference in theirentirety), araB promoter (Better et al., Science (1988) 240, 1041-1043,which is incorporated herein by reference in its entirety), or T7promoter. Examples of such vectors include the vectors mentioned aboveas well as pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system”(manufactured by Qiagen N. V.), pEGFP, and pET (in this case, the hostis preferably BL21 expressing T7 RNA polymerase).

The vectors may contain a signal sequence for polypeptide secretion. Inthe case of production in the periplasm of E. coli, pelB signal sequence(Lei, S. P. et al., J. Bacteriol. (1987) 169, 4397, which isincorporated herein by reference in its entirety) can be used as thesignal sequence for polypeptide secretion. The vectors can betransferred to the host cells by use of, for example, a Lipofectinmethod, a calcium phosphate method, or a DEAE-dextran method.

In addition to the expression vectors for E. coli, examples of thevectors for producing the polypeptide of the present invention includemammal-derived expression vectors (e.g., pcDNA3 (manufactured byInvitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p. 5322,which is incorporated herein by reference in its entirety), pEF, andpCDM8), insect cell-derived expression vectors (e.g., “Bac-to-BACbaculovirus expression system” (manufactured by GIBCO BRL), andpBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2),animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw),retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derivedexpression vectors (e.g., “Pichia Expression Kit” (manufactured byInvitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis-derivedexpression vectors (e.g., pPL608 and pKTH50).

For the purpose of expression in animal cells such as CHO cells, COScells, NIH3T3 cells, or HEK293 cells, the vectors indispensably have apromoter necessary for intracellular expression, for example, SV40promoter (Mulligan et al., Nature (1979) 277, 108, which is incorporatedherein by reference in its entirety), MMTV-LTR promoter, EF1α promoter(Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which isincorporated herein by reference in its entirety), CAG promoter (Gene.(1991) 108, 193, which is incorporated herein by reference in itsentirety), or CMV promoter and, more preferably, have a gene forscreening for transformed cells (e.g., a drug resistance gene that canwork as a marker by a drug (neomycin, G418, etc.)). Examples of thevectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV,pOPRSV, and pOP13. In addition, EBNA1 protein may be coexpressedtherewith for the purpose of increasing the number of gene copies. Inthis case, vectors having a replication origin OriP are used (BiotechnolBioeng. 2001 Oct. 20; 75 (2): 197-203; and Biotechnol Bioeng. 2005 Sep.20; 91 (6): 670-7).

An exemplary method intended to stably express the gene and increase thenumber of intracellular gene copies involves transforming CHO cellsdeficient in nucleic acid synthesis pathway with vectors having a DHFRgene serving as a complement thereto (e.g., pCHOI) and usingmethotrexate (MTX) in the gene amplification. An exemplary methodintended to transiently express the gene involves using COS cells havingan SV40 T antigen gene on their chromosomes to transform the cells withvectors having a replication origin of SV40 (pcD, etc.). A replicationorigin derived from polyomavirus, adenovirus, bovine papillomavirus(BPV), or the like can also be used. In order to increase the number ofgene copies in the host cell system, the expression vectors can containa selective marker such as an aminoglycoside phosphotransferase (APH)gene, a thymidine kinase (TK) gene, an E. coli xanthine guaninephosphoribosyltransferase (Ecogpt) gene, or a dihydrofolate reductase(dhfr) gene.

The antibody can be recovered, for example, by culturing the transformedcells and then separating the antibody from within themolecule-transformed cells or from the culture solution thereof. Theantibody can be separated and purified by appropriately using incombination methods such as centrifugation, ammonium sulfatefractionation, salting out, ultrafiltration, C1q, FcRn, protein A andprotein G columns, affinity chromatography, ion-exchangedchromatography, and gel filtration chromatography.

The technique mentioned above, such as the knobs-into-holes technology(WO1996/027011; Ridgway J B et al., Protein Engineering (1996) 9,617-621; and Merchant A M et al., Nature Biotechnology (1998) 16,677-681) or the technique of suppressing the unintended associationbetween H chains by the introduction of electric charge repulsion(WO2006/106905), can be applied to a method for efficiently preparingthe multispecific antibody.

The present invention further provides a method for producing theantigen-binding molecule of the present invention and specificallyprovides a method for producing an antigen-binding molecule comprising:an antibody variable region that is capable of binding to two differentantigens (first antigen and second antigen), but does not bind to thefirst antigen and the second antigen at the same time (this variableregion is referred to as a first variable region); and a variable regionbinding to a third antigen different from the first antigen and thesecond antigen (this variable region is referred to as a second variableregion), the method comprising the step of preparing an antigen-bindingmolecule library containing diverse amino acid sequences of the firstvariable region.

Examples thereof can include a production method comprising thefollowing steps:

(i) preparing a library of antigen-binding molecules with at least oneamino acid altered in their antibody variable regions each binding tothe first antigen or the second antigen, wherein the altered variableregions differ in at least one amino acid from each other;(ii) selecting, from the prepared library, an antigen-binding moleculecomprising a variable region that has binding activity against the firstantigen and the second antigen, but does not bind to the first antigenand the second antigen at the same time;(iii) culturing a host cell comprising a nucleic acid encoding thevariable region of the antigen-binding molecule selected in the step(ii), and a nucleic acid encoding a variable region of anantigen-binding molecule binding to the third antigen, to express anantigen-binding molecule comprising the antibody variable region that iscapable of binding to the first antigen and the second antigen, but doesnot bind to the first antigen and the second antigen at the same time,and the variable region binding to the third antigen; and(iv) recovering the antigen-binding molecule from the host cellcultures.

In this production method, the step (ii) may be the following selectionstep:

(v) selecting, from the prepared library, an antigen-binding moleculecomprising a variable region that has binding activity against the firstantigen and the second antigen, but does not bind to the first antigenand the second antigen each expressed on a different cell, at the sametime.

The antigen-binding molecules used in the step (i) are not particularlylimited as long as these molecules each comprise an antibody variableregion. The antigen-binding molecules may be antibody fragments such asFv, Fab, or Fab′ or may be Fc region-containing antibodies.

The amino acid to be altered is selected from, for example, amino acidswhose alteration does not cancel the binding to the antigen, in theantibody variable region binding to the first antigen or the secondantigen.

In the present invention, one amino acid alteration may be used alone,or a plurality of amino acid alterations may be used in combination.

In the case of using a plurality of amino acid alterations incombination, the number of the alterations to be combined is notparticularly limited and is, for example, 2 or more and 30 or less,preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or moreand 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 ormore and 5 or less, or 2 or more and 3 or less.

The plurality of amino acid alterations to be combined may be added toonly the antibody heavy chain variable domain or light chain variabledomain or may be appropriately distributed to both of the heavy chainvariable domain and the light chain variable domain.

Examples of the region preferred for the amino acid alteration includesolvent-exposed regions and loops in the variable region. Among others,CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabatnumbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the Hchain variable domain and Kabat numbering positions 24 to 34, 50 to 56,and 89 to 97 in the L chain variable domain are preferred. Kabatnumbering positions 31, 52a to 61, 71 to 74, and 97 to 101 in the Hchain variable domain and Kabat numbering positions 24 to 34, 51 to 56,and 89 to 96 in the L chain variable domain are more preferred.

The alteration of an amino acid residue also include: the randomalteration of amino acids in the region mentioned above in the antibodyvariable region binding to the first antigen or the second antigen; andthe insertion of a peptide previously known to have binding activityagainst the desired antigen, to the region mentioned above. Theantigen-binding molecule of the present invention can be obtained byselecting a variable region that is capable of binding to the firstantigen and the second antigen, but cannot bind to these antigens at thesame time, from among the antigen-binding molecules thus altered.Examples of the peptide previously known to have binding activityagainst the desired antigen include peptides shown in Table 1 above.

Whether the variable region is capable of binding to the first antigenand the second antigen, but cannot bind to these antigens at the sametime, and further, whether the variable region is capable of binding toboth the first antigen and the second antigen at the same time when anyone of the first antigen and the second antigen resides on a cell andthe other antigen exists alone, both of the antigens each exist alone,or both of the antigens reside on the same cell, but cannot bind tothese antigens each expressed on a different cell, at the same time, canalso be confirmed according to the method mentioned above.

The present invention further provides a method for producing theantigen-binding molecule of the present invention and specificallyprovides a method for producing an antigen-binding molecule comprisingan antibody variable region that is capable of binding to two differentantigens (first antigen and second antigen), but does not bind to thefirst antigen and the second antigen at the same time (this variableregion is referred to as a first variable region), the method comprisingthe step of preparing an antigen-binding molecule library containingdiverse amino acid sequences of the first variable region.

Examples of the method for producing such an antigen-binding moleculecan include a production method comprising the following steps:

(i) preparing a library of antigen-binding molecules with at least oneamino acid altered in their antibody variable regions each binding tothe first antigen or the second antigen, wherein the altered variableregions differ in at least one amino acid from each other;(ii) selecting, from the prepared library, an antigen-binding moleculecomprising a variable region that has binding activity against the firstantigen and the second antigen, but does not bind to the first antigenand the second antigen at the same time;(iii) culturing a host cell comprising a nucleic acid encoding thevariable region of the antigen-binding molecule selected in the step(ii), to express an antigen-binding molecule comprising the antibodyvariable region that is capable of binding to the first antigen and thesecond antigen, but does not bind to the first antigen and the secondantigen at the same time; and(iv) recovering the antigen-binding molecule from the host cellcultures.

Examples of the region preferred for the amino acid alteration include aheavy chain variable domain. More preferred examples thereof includesolvent-exposed regions and loops in the variable domain. Among others,CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabatnumbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the Hchain variable domain and Kabat numbering positions 24 to 34, 50 to 56,and 89 to 97 in the L chain variable domain are preferred. Kabatnumbering positions 31, 52a to 61, 71 to 74, and 97 to 101 in the Hchain variable domain and Kabat numbering positions 24 to 34, 51 to 56,and 89 to 96 in the L chain variable domain are more preferred.

In this production method, the step (ii) may be the following selectionstep:

(v) selecting, from the prepared library, an antigen-binding moleculecomprising a variable region that has binding activity against the firstantigen and the second antigen, but does not bind to the first antigenand the second antigen each expressed on a different cell, at the sametime.

The antigen-binding molecules used in the step (i) are not particularlylimited as long as these molecules each comprise an antibody variableregion. The antigen-binding molecules may be antibody fragments such asFv, Fab, or Fab′ or may be Fc region-containing antibodies.

The amino acid to be altered is selected from, for example, amino acidswhose alteration does not cancel the binding to the antigen, in theantibody variable region binding to the first antigen or the secondantigen.

In the present invention, one amino acid alteration may be used alone,or a plurality of amino acid alterations may be used in combination.

In the case of using a plurality of amino acid alterations incombination, the number of the alterations to be combined is notparticularly limited and is, for example, 2 or more and 30 or less,preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or moreand 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 ormore and 5 or less, or 2 or more and 3 or less.

The plurality of amino acid alterations to be combined may be added onlyto either the antibody heavy chain variable domain or light chainvariable domain or may be appropriately distributed to both of the heavychain variable domain and the light chain variable domain.

The alteration of an amino acid residue also include: the randomalteration of amino acids in the region mentioned above in the antibodyvariable region binding to the first antigen or the second antigen; andthe insertion of a peptide previously known to have binding activityagainst the desired antigen, to the region mentioned above. Theantigen-binding molecule of the present invention can be obtained byselecting a variable region that is capable of binding to the firstantigen and the second antigen, but cannot bind to these antigens at thesame time, from among the antigen-binding molecules thus altered.Examples of the peptide previously known to have binding activityagainst the desired antigen include peptides shown in Table 1 above.

Whether the variable region is capable of binding to the first antigenand the second antigen, but cannot bind to these antigens at the sametime, and further, whether the variable region is capable of binding toboth the first antigen and the second antigen at the same time when anyone of the first antigen and the second antigen resides on a cell andthe other antigen exists alone, both of the antigens each exist alone,or both of the antigens reside on the same cell, but cannot bind tothese antigens each expressed on a different cell, at the same time, canalso be confirmed according to the method mentioned above.

The antigen-binding molecule produced by any of these production methodsis also included in the present invention.

The type or range of the amino acid alteration introduced by the methodof the present invention is not particularly limited.

In a non-limiting embodiment, examples of the library of the presentinvention include a library consisting of antigen-binding moleculesbinding to CD3 (in the case of human CD3, γ chain, δ chain, or ε chainconstituting the human CD3) selected as the first antigen, and anarbitrary second antigen.

In the present specification, the “library” refers to a plurality ofantigen-binding molecules or a plurality of fusion polypeptidescomprising the antigen-binding molecules, or nucleic acids orpolynucleotides encoding these sequences. The plurality ofantigen-binding molecules or the plurality of fusion polypeptidescomprising the antigen-binding molecules, included in the library areantigen-binding molecules differing in sequence from each other, nothaving single sequences, or fusion polypeptides comprising theantigen-binding molecules.

In one embodiment of the present invention, a fusion polypeptide of theantigen-binding molecule of the present invention and a heterologouspolypeptide can be prepared. In one embodiment, the fusion polypeptidecan comprise the antigen-binding molecule of the present invention fusedwith at least a portion of a viral coat protein selected from the groupconsisting of, for example, viral coat proteins pIII, pVIII, pVII, pIX,Soc, Hoc, gpD, and pVI, and variants thereof.

In one embodiment, the antigen-binding molecule of the present inventioncan be ScFv, a Fab fragment, F(ab)₂, or F(ab′)₂. In another embodiment,the present invention provides a library consisting essentially of aplurality of fusion polypeptides differing in sequence from each other,the fusion polypeptides each comprising any of these antigen-bindingmolecules and a heterologous polypeptide. Specifically, the presentinvention provides a library consisting essentially of a plurality offusion polypeptides differing in sequence from each other, the fusionpolypeptides each comprising any of these antigen-binding moleculesfused with at least a portion of a viral coat protein selected from thegroup consisting of, for example, viral coat proteins pIII, pVIII, pVII,pIX, Soc, Hoc, gpD, and pVI, and variants thereof. The antigen-bindingmolecule of the present invention may further comprise a dimerizationdomain. In one embodiment, the dimerization domain can be locatedbetween the antibody heavy chain or light chain variable domain and atleast a portion of the viral coat protein. This dimerization domain maycomprise at least one dimerization sequence and/or a sequence comprisingone or more cysteine residues. This dimerization domain can bepreferably linked to the C terminus of the heavy chain variable domainor constant domain. The dimerization domain can assume variousstructures, depending on whether the antibody variable domain isprepared as a fusion polypeptide component with the viral coat proteincomponent (an amber stop codon following the dimerization domain isabsent) or depending on whether the antibody variable domain is preparedpredominantly without comprising the viral coat protein component (e.g.,an amber stop codon following the dimerization domain is present). Whenthe antibody variable domain is prepared predominantly as a fusionpolypeptide with the viral coat protein component, bivalent display isbrought about by one or more disulfide bonds and/or a singledimerization sequence.

The term “differing in sequence from each other” in a plurality ofantigen-binding molecules differing in sequence from each other asdescribed herein means that the individual antigen-binding molecules inthe library have distinct sequences. Specifically, the number of thedistinct sequences in the library reflects the number of independentclones differing in sequences in the library and may also be referred toas a “library size”. The library size of a usual phage display libraryis 10⁶ to 10¹² and can be expanded to 10′ by the application of atechnique known in the art such as a ribosome display method. The actualnumber of phage particles for use in panning selection for the phagelibrary, however, is usually 10 to 10,000 times larger than the librarysize. This excessive multiple, also called the “number of equivalents ofthe library”, represents that 10 to 10,000 individual clones may havethe same amino acid sequence. Accordingly, the term “differing insequence from each other” described in the present invention means thatthe individual antigen-binding molecules in the library excluding thenumber of equivalents of the library have distinct sequences and morespecifically means that the library has 10⁶ to 10¹⁴, preferably 10⁷ to10¹², more preferably 10⁸ to 10¹¹, particularly preferably 10⁸ to 10¹⁰antigen-binding molecules differing in sequence from each other.

The term “consisting essentially of” in the library consistingessentially of a plurality of antigen-binding molecules as described inthe present invention reflects the number of antigen-binding moleculesdiffering in binding activity against the first and/or second antigen,among the independent clones differing in sequence in the library.Specifically, the library preferably has at least 10⁴ antigen-bindingmolecules that exhibit such binding activity. More preferably, thepresent invention provides the library having at least 10⁵antigen-binding molecules that exhibit such binding activity. Furtherpreferably, the present invention provides the library having at least10⁶ antigen-binding molecules that exhibit such binding activity.Particularly preferably, the present invention provides the libraryhaving at least 10⁷ antigen-binding molecules that exhibit such bindingactivity. Also preferably, the present invention provides the libraryhaving at least 10⁸ antigen-binding molecules that exhibit such bindingactivity. In other words, the term may be preferably indicated by theratio of the number of the antigen-binding molecules differing inbinding activity against the first and/or second antigen to the numberof the independent clones differing in sequence in the library.Specifically, the present invention provides the library comprisingantigen-binding molecules that exhibit such binding activity at a ratioof 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%,further preferably 2% to 20%, particularly preferably 4% to 10% to thenumber of the independent clones differing in sequence in the library.Fusion polypeptides, polynucleotide molecules, or vectors can also beindicated by the number of molecules or the ratio to all molecules, asin the above case. Likewise, viruses can also be indicated by the numberof virus individuals or the ratio to all individuals, as in the abovecase.

The “phage display” as described herein refers to an approach by whichvariant polypeptides are displayed as fusion proteins with at least aportion of coat proteins on the particle surface of phages, for example,filamentous phages. The phage display is useful because a large libraryof randomized protein variants can be rapidly and efficiently screenedfor a sequence binding to a target antigen with high affinity. Thedisplay of peptide and protein libraries on the phages has been used forscreening millions of polypeptides for ones with specific bindingproperties. A polyvalent phage display method has been used fordisplaying small random peptides and small proteins through fusion withfilamentous phage gene III or gene VIII (Wells and Lowman, Curr. Opin.Struct. Biol. (1992) 3, 355-362; and references cited therein).Monovalent phage display involves fusing a protein or peptide library togene III or a portion thereof, and expressing fusion proteins at lowlevels in the presence of wild-type gene III protein so that each phageparticle displays one copy or none of the fusion proteins. Themonovalent phages have a lower avidity effect than that of thepolyvalent phages and are therefore screened on the basis of endogenousligand affinity using phagemid vectors, which simplify DNA manipulation(Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991)3, 205-216).

The “phagemid” refers to a plasmid vector having a bacterial replicationorigin, for example, ColE1, and a copy of an intergenic region of abacteriophage. A phagemid derived from any bacteriophage known in theart, for example, a filamentous bacteriophage or a lambdoidbacteriophage, can be appropriately used. Usually, the plasmid alsocontains a selective marker for antibiotic resistance. DNA fragmentscloned into these vectors can grow as plasmids. When cells harboringthese vectors possess all genes necessary for the production of phageparticles, the replication pattern of plasmids is shifted to rollingcircle replication to form copies of one plasmid DNA strand and packagephage particles. The phagemid can form infectious or non-infectiousphage particles. This term includes a phagemid comprising a phage coatprotein gene or a fragment thereof bound with a heterologous polypeptidegene by gene fusion such that the heterologous polypeptide is displayedon the surface of the phage particle.

The term “phage vector” means a double-stranded replicativebacteriophage that comprises a heterologous gene and is capable ofreplicating. The phage vector has a phage replication origin thatpermits phage replication and phage particle formation. The phage ispreferably a filamentous bacteriophage, for example, an M13, f1, fd, orPf3 phage or a derivative thereof, or a lambdoid phage, for example,lambda, 21, phi80, phi81, 82, 424, 434, or any other phage or aderivative thereof.

The term “oligonucleotide” refers to a short single- or double-strandedpolydeoxynucleotide that is chemically synthesized by a method known inthe art (e.g., phosphotriester, phosphite, or phosphoramidite chemistryusing a solid-phase approach such as an approach described in EP266032;or a method via deoxynucleotide H-phosphonate intermediates described inFroeshler et al., Nucl. Acids. Res. (1986) 14, 5399-5407). Other methodsfor oligonucleotide synthesis include the polymerase chain reactiondescribed below and other autoprimer methods and oligonucleotidesyntheses on solid supports. All of these methods are described inEngels et al., Agnew. Chem. Int. Ed. Engl. (1989) 28, 716-734. Thesemethods are used if the whole nucleic acid sequence of the gene is knownor if a nucleic acid sequence complementary to the coding strand isavailable. Alternatively, a possible nucleic acid sequence may beappropriately predicted using known and preferred residues encoding eachamino acid residue, if the target amino acid sequence is known. Theoligonucleotide can be purified using polyacrylamide gels or molecularsizing columns or by precipitation.

The terms “fusion protein” and “fusion polypeptide” refer to apolypeptide having two segments covalently linked to each other. Thesesegments in the polypeptide differ in character. This character may be,for example, a biological property such as in vitro or in vivo activity.Alternatively, this character may be a single chemical or physicalproperty, for example, binding to a target antigen or catalysis ofreaction. These two segments may be linked either directly through asingle peptide bond or via a peptide linker containing one or more aminoacid residues. Usually, these two segments and the linker are located inthe same reading frame. Preferably, the two segments of the polypeptideare obtained from heterologous or different polypeptides.

The term “coat protein” refers to a protein, at least a portion of whichis present on the surface of a viral particle. From a functionalstandpoint, the coat protein is an arbitrary protein that binds to viralparticles in the course of construction of viruses in host cells andremains bound therewith until viral infection of other host cells. Thecoat protein may be a major coat protein or may be a minor coat protein.The minor coat protein is usually a coat protein present in viral capsidat preferably at least approximately 5, more preferably at leastapproximately 7, further preferably at least approximately 10 or moreprotein copies per virion. The major coat protein can be present attens, hundreds, or thousands of copies per virion. Examples of the majorcoat protein include filamentous phage p8 protein.

In a non-limiting embodiment of the present invention, examples of amethod for preparing the library include the following 6 methods:

1. a method which involves inserting a peptide (this term is used toinclude a polypeptide and a protein) binding to the second antigen toantigen-binding molecules each binding to the first antigen;2. a method which involves preparing a library such that various aminoacids appear positions that permit alteration to a larger length(extension) of loops in antigen-binding molecules, and obtaining anantigen-binding molecule having binding activity against an arbitrarysecond antigen from the library by using the binding activity againstthe antigen as an index;3. a method which involves identifying amino acids that maintain bindingactivity against the first antigen by use of an antibody prepared bysite-directed mutagenesis from an antigen-binding molecule previouslyknown to bind to the first antigen, and obtaining an antigen-bindingmolecule having binding activity against an arbitrary second antigenfrom a library in which the identified amino acids appear by using thebinding activity against the antigen as an index;4. the method 3 which further involves preparing an antibody librarysuch that various amino acids appear positions that permit alteration toa larger length (extension) of loops in antigen-binding molecules, andobtaining an antigen-binding molecule having binding activity against anarbitrary second antigen from the library by using the binding activityagainst the antigen as an index;5. the method 1, 2, 3, or 4 which further involves altering theantigen-binding molecules such that glycosylation sequences (e.g., NxSand NxT wherein x is an amino acid other than P) appear to add theretosugar chains that are recognized by sugar chain receptors (e.g.,high-mannose-type sugar chains are added thereto and thereby recognizedby high-mannose receptors; it is known that the high-mannose-type sugarchains are obtained by the addition of kifunensine at the time ofantibody expression (mAbs. 2012 July-August; 4 (4): 475-87)); and6. the method 1, 2, 3, or 4 which further involves adding theretodomains each binding to the second antigen through a covalent bond byinserting Cys, Lys, or a non-natural amino acid to loops or sites foundto be alterable to various amino acids or substituting these sites withCys, Lys, or a non-natural amino acid (this method is typified byantibody drug conjugates and is a method for conjugation to Cys, Lys, ora non-natural amino acid through a covalent bond (mAbs 6: 1, 34-45;January/February 2014; WO2009/134891 A2; and Bioconjug Chem. 2014 Feb.19; 25 (2): 351-61)).

In these 6 library preparation methods listed above, the amino acidsubstitution site in each antigen-binding molecule or the peptideinsertion site in the antigen-binding molecule is preferably a site in aFab or variable region in the antigen-binding molecule. Examples of thepreferred region include solvent-exposed regions and loops in thevariable region. Among others, CDR1, CDR2, CDR3, FR3, and loops arepreferred. Specifically, Kabat numbering positions 31 to 35, 50 to 65,71 to 74, and 95 to 102 in the H chain variable domain and Kabatnumbering positions 24 to 34, 50 to 56, and 89 to 97 in the L chainvariable domain are preferred. Kabat numbering positions 31, 52a to 61,71 to 74, and 97 to 101 in the H chain variable domain and Kabatnumbering positions 24 to 34, 51 to 56, and 89 to 96 in the L chainvariable domain are more preferred.

In one embodiment, examples of the method 1 which involves inserting apeptide binding to the second antigen to antigen-binding molecules eachbinding to the first antigen can also include a method of insertingG-CSF as exemplified in Angew Chem Int Ed Engl. 2013 Aug. 5; 52 (32):8295-8. In another embodiment, the peptide to be inserted can beobtained from a peptide-displaying library. Alternatively, the whole ora portion of a naturally occurring protein may be used.

In order to identify amino acids that maintain binding activity againsta first antigen CD3 (in the case of human CD3, γ chain, 8 chain, or cchain constituting the human CD3), for example, one-amino acidalteration antibodies are prepared by amino acid alteration at sitespresumed to participate in antigen binding, and these antibodies can beexamined. The CD3 binding of the one-amino acid alteration antibodiescan be evaluated by an appropriately selected method generally known tothose skilled in the art and can be determined by, for example, ELISA,FACS (fluorescence activated cell sorting), ALPHAScreen (amplifiedluminescent proximity homogeneous assay screen), or the BIACORE methodbased on a surface plasmon resonance (SPR) phenomenon.

In order to identify the amino acids that maintain binding activityagainst CD3, results about the ratios of the amounts of, for example,various altered forms, bound to the amount of the correspondingunaltered antibody bound can be used. Specifically, when the amount ofthe corresponding unaltered antibody bound is defined as X and theamount of the one-amino acid altered form bound is defined as Y, a valueof Z (ratio of amounts bound)=Y/X can be used. The altered form can beconsidered to maintain the binding relative to the correspondingunaltered antibody when Z (ratio of amount bound) is 0.5 or more, 0.6 ormore, 0.7 or more, 0.8 or more, or 0.9 or more, preferably 0.8 or more.The antibody library can be prepared such that such amino acids thatmaintain binding appear.

ECM (extracellular matrix) is an extracellular constituent and residesat various sites in vivo. Therefore, an antibody strongly binding to ECMis known to have poorer kinetics in blood (shorter half-life)(WO2012093704 A1). Thus, amino acids that do not enhance ECM binding arepreferably selected as the amino acids that appear in the antibodylibrary.

In order to select the amino acids that do not enhance ECM binding, theECM binding is evaluated, for example, according to a method ofReference Example 2. The ECM binding value (ECL reaction) of eachaltered form is divided by the ECM binding value of an antibody MRA (Hchain: SEQ ID NO: 57, L chain: SEQ ID NO: 58), and the resulting valuecan be used. An effective value up to 5 times, 6 times, 7 times, 8times, 9 times, 10 times, 15 times, 20 times, or 30 times can be adoptedas this value in consideration of the effect of enhancing ECM binding bya plurality of alterations. Preferably, an effective value up to 10times can be adopted to the library. The antibody library can beprepared such that the amino acids thus selected appear.

In the case of inserting, but not limited to, a peptide of 6 amino acidsto CDR3, the binding to ECM is enhanced if the extended loop of CDR3 isrich in amino acids having a positively charged side chain. Therefore,it is preferred that three or more amino acids having a positivelycharged side chain should not appear in the loop.

In the library of the present invention, a peptide for enhancing itsdiversity can be inserted to each variable region. Examples of theregion preferred for the peptide insertion include solvent-exposedregions and loops in the variable region. Among others, CDR1, CDR2,CDR3, FR3, and loops are preferred. Specifically, Kabat numberingpositions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the H chainvariable domain and Kabat numbering positions 24 to 34, 50 to 56, and 89to 97 in the L chain variable domain are preferred. Kabat numberingpositions 31, 52a to 61, 71 to 74, and 97 to 101 in the H chain variabledomain and Kabat numbering positions 24 to 34, 51 to 56, and 89 to 96 inthe L chain variable domain are more preferred. A region of Kabatnumbering positions 99 and 100 in the H chain variable domain is furtherpreferred. Also, an amino acid that increases antigen-binding activitymay be further introduced at the time of the amino acid alteration.

In a non-limiting embodiment of the present invention, examples of thelength of the peptide to be inserted include 1 to 3 amino acids, 4 to 6amino acids, 7 to 9 amino acids, 10 to 12 amino acids, 13 to 15 aminoacids, 15 to 20 amino acids, and 21 to 25 amino acids. The length of thepeptide to be inserted is preferably 1 to 3 amino acids, 4 to 6 aminoacids, or 7 to 9 amino acids.

The insertion site and the length of the peptide for enhancing thediversity of the library can be studied by preparing peptide-insertedmolecules and evaluating the CD3 binding of the molecules. The CD3binding can be evaluated by an appropriately selected method generallyknown to those skilled in the art and can be determined by, for example,ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen(amplified luminescent proximity homogeneous assay screen), or theBIACORE method based on a surface plasmon resonance (SPR) phenomenon.

In a non-limiting embodiment of the present invention, an antibodylibrary for obtaining an antibody binding to CD3 and the second antigencan be designed as follows:

step 1: selecting amino acids that maintain the ability to bind to CD3(to secure 80% or more of the amount of the unaltered antibody bound toCD3).

The library for obtaining an antibody binding to CD3 and the secondantigen can be prepared, for example, such that the amino acids selectedin the step 1 appear.

In a non-limiting embodiment of the present invention, the antibodylibrary for obtaining an antibody binding to CD3 and the second antigencan be designed as follows:

step 1: selecting amino acids that maintain the ability to bind to CD3(to secure 80% or more of the amount of the unaltered antibody bound toCD3); andstep 2: inserting an amino acid to between positions 99 and 100 (Kabatnumbering) in H chain CDR3.

The library for obtaining an antibody binding to CD3 and the secondantigen can be prepared with the library diversity enhanced, forexample, by the step 1 as well as amino acid insertion to the CDR3domain in the step 2.

In a non-limiting embodiment of the present invention, the antibodylibrary for obtaining an antibody binding to CD3 and the second antigencan be designed as follows:

step 1: selecting amino acids that maintain the ability to bind to CD3(to secure 80% or more of the amount of the unaltered antibody bound toCD3);step 2: selecting amino acids that keep ECM binding within 10 times thatof MRA compared with before alteration; andstep 3: inserting an amino acid to between positions 99 and 100 (Kabatnumbering) in H chain CDR3.

The amino acids that do not enhance ECM binding can also be selected asthe amino acids appearing in the library, for example, by the steps 1and 3 as well as the step 2, though the present invention is not limitedby this approach. Even library design without the step 2 allows anantigen-binding molecule obtained from the library to be assayed andevaluated for ECM binding.

In a non-limiting embodiment of the present invention, a VH domainCE115HA000 (SEQ ID NO: 52) may be used as a template sequence for CD3(CD3ε)-binding antibody. In such a case, examples of amino acids for usein library design can include any one or more of amino acids at Kabatnumbering positions 11, 31, 52a, 52b, 52c, 53, 54, 56, 57, 61, 72, 78,98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, and 101 containedin the heavy chain variable domain.

For the library, it is preferred to introduce amino acid alterationV11L/L78I to the VH domain CE115HA000 (SEQ ID NO: 52), though thelibrary according to the present invention is not limited thereto. Forthe library, it is further preferred to introduce amino acid alterationV11L/D72A/L78I/D101Q to the VH domain CE115HA000 (SEQ ID NO: 52), thoughthe library according to the present invention is not limited thereto.

In a non-limiting embodiment of the present invention, a VL domainGLS3000 (SEQ ID NO: 53) may be used as a template sequence for CD3(CD3ε)-binding antibody. In such a case, examples of amino acids for usein library design can include any one or more of amino acids at Kabatnumbering positions 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34,51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, 96, and 107contained in the light chain variable domain.

The library design according to the present invention includes, but isnot particularly limited to, the design of a library comprisingantigen-binding domains with an amino acid at a particular site alteredto the desired amino acid, or a plurality of altered forms ofantigen-binding molecules comprising antigen-binding domains by use of alibrary technique known in the art, for example, NNK or TRIM Library(Gonzalez-Munoz A et al., mAbs 2012; Lee CV et al., J Mol Biol. 2004;Knappik A. et al., J Mol Biol. 2000; and Tiller T et al., mAbs 2013).

In the present invention, the term “one or more amino acids” is notlimited to a particular number of amino acids and may be 2 or more typesof amino acids, 5 or more types of amino acids, 10 or more types ofamino acids, 15 or more types of amino acids, or 20 types of aminoacids.

As for fusion polypeptide display, the fusion polypeptide of thevariable region of the antigen-binding molecule can be displayed invarious forms on the surface of cells, viruses, or phagemid particles.These forms include single-chain Fv fragments (scFvs), F(ab) fragments,and multivalent forms of these fragments. The multivalent forms arepreferably ScFv, Fab, and F(ab′) dimers, which are referred to as(ScFv)2, F(ab)2, and F(ab′)2, respectively, herein. The display of themultivalent forms is preferred, probably in part because the displayedmultivalent forms usually permit identification of low-affinity clonesand/or have a plurality of antigen-binding sites that permit moreefficient selection of rare clones in the course of selection.

Methods for displaying fusion polypeptides comprising antibody fragmentson the surface of bacteriophages are known in the art and described in,for example, WO1992001047 and the present specification. Other relatedmethods are described in WO1992020791, WO1993006213, WO1993011236, and1993019172. Those skilled in the art can appropriately use thesemethods. Other public literatures (H.R. Hoogenboom & G. Winter (1992) J.Mol. Biol. 227, 381-388, WO1993006213, and WO1993011236) disclose theidentification of antibodies using artificially rearranged variableregion gene repertoires against various antigens displayed on thesurface of phages.

In the case of constructing a vector for display in the form of scFv,this vector comprises nucleic acid sequences encoding the light chainvariable domain and the heavy chain variable domain of theantigen-binding molecule. In general, the nucleic acid sequence encodingthe heavy chain variable domain of the antigen-binding molecule is fusedwith a nucleic acid sequence encoding a viral coat protein constituent.The nucleic acid sequence encoding the light chain variable domain ofthe antigen-binding molecule is linked to the heavy chain variabledomain nucleic acid of the antigen-binding molecule through a nucleicacid sequence encoding a peptide linker. The peptide linker generallycontains approximately 5 to 15 amino acids. Optionally, an additionalsequence encoding, for example, a tag useful in purification ordetection, may be fused with the 3′ end of the nucleic acid sequenceencoding the light chain variable domain of the antigen-binding moleculeor the nucleic acid sequence encoding the heavy chain variable domain ofthe antigen-binding molecule, or both.

In the case of constructing a vector for display in the form of F(ab),this vector comprises nucleic acid sequences encoding the variabledomains of the antigen-binding molecule and the constant domains of theantigen-binding molecule. The nucleic acid sequence encoding the lightchain variable domain is fused with the nucleic acid sequence encodingthe light chain constant domain. The nucleic acid sequence encoding theheavy chain variable domain of the antigen-binding molecule is fusedwith the nucleic acid sequence encoding the heavy chain constant CH1domain. In general, the nucleic acid sequence encoding the heavy chainvariable domain and constant domain is fused with a nucleic acidsequence encoding the whole or a portion of a viral coat protein. Theheavy chain variable domain and constant domain are preferably expressedas a fusion product with at least a portion of the viral coat protein,while the light chain variable domain and constant domain are expressedseparately from the heavy chain-viral coat fusion protein. The heavychain and the light chain may be associated with each other through acovalent bond or a non-covalent bond. Optionally, an additional sequenceencoding, for example, a polypeptide tag useful in purification ordetection, may be fused with the 3′ end of the nucleic acid sequenceencoding the light chain constant domain of the antigen-binding moleculeor the nucleic acid sequence encoding the heavy chain constant domain ofthe antigen-binding molecule, or both.

As for vector transfer to host cells, the vectors constructed asdescribed above are transferred to host cells for amplification and/orexpression. The vectors can be transferred to host cells by atransformation method known in the art, including electroporation,calcium phosphate precipitation, and the like. When the vectors areinfectious particles such as viruses, the vectors themselves invade thehost cells. Fusion proteins are displayed on the surface of phageparticles by the transfection of host cells with replicable expressionvectors having inserts of polynucleotides encoding the fusion proteinsand the production of the phage particles by an approach known in theart.

The replicable expression vectors can be transferred to host cells byuse of various methods. In a non-limiting embodiment, the vectors can betransferred to the cells by electroporation as described inWO2000106717. The cells are cultured at 37° C., optionally forapproximately 6 to 48 hours (or until OD at 600 nm reaches 0.6 to 0.8)in a standard culture medium. Next, the culture medium is centrifuged,and the culture supernatant is removed (e.g., by decantation). At theinitial stage of purification, the cell pellet is preferably resuspendedin a buffer solution (e.g., 1.0 mM HEPES (pH 7.4)). Next, the suspensionis centrifuged again to remove the supernatant. The obtained cell pelletis resuspended in glycerin diluted to, for example, 5 to 20% V/V. Thesuspension is centrifuged again for the removal of the supernatant toobtain cell pellet. The cell pellet is resuspended in water or dilutedglycerin. On the basis of the measured cell density of the resultingsuspension, the final cell density is adjusted to a desired densityusing water or diluted glycerin.

Examples of preferred recipient cells include an E. coli strain SS320capable of responding to electroporation (Sidhu et al., Methods Enzymol.(2000) 328, 333-363). The E. coli strain SS320 has been prepared by thecoupling of MC1061 cells with XL1-BLUE cells under conditions sufficientfor transferring fertility episome (F′ plasmid) or XL1-BLUE into theMC1061 cells. The E. coli strain SS320 has been deposited with ATCC(10801 University Boulevard, Manassas, Va.) under deposition No. 98795.Any F′ episome that permits phage replication in this strain can be usedin the present invention. Appropriate episome may be obtained fromstrains deposited with ATCC or may be obtained as a commerciallyavailable product (TG1, CJ236, CSH18, DHF′, ER2738, JM101, JM103, JM105,JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

Use of higher DNA concentrations (approximately 10 times) inelectroporation improves transformation frequency and increases theamount of DNAs transforming the host cells. Use of high cell densitiesalso improves the efficiency (approximately 10 times). The increasedamount of transferred DNAs can yield a library having greater diversityand a larger number of independent clones differing in sequence. Thetransformed cells are usually selected on the basis of the presence orabsence of growth on a medium containing an antibiotic.

The present invention further provides a nucleic acid encoding theantigen-binding molecule of the present invention. The nucleic acid ofthe present invention may be in any form such as DNA or RNA.

The present invention further provides a vector comprising the nucleicacid of the present invention. The type of the vector can beappropriately selected by those skilled in the art according to hostcells that receive the vector. For example, any of the vectors mentionedabove can be used.

The present invention further relates to a host cell transformed withthe vector of the present invention. The host cell can be appropriatelyselected by those skilled in the art. For example, any of the host cellsmentioned above can be used.

The present invention also provides a pharmaceutical compositioncomprising the antigen-binding molecule of the present invention and apharmaceutically acceptable carrier. The pharmaceutical composition ofthe present invention can be formulated according to a method known inthe art by supplementing the antigen-binding molecule of the presentinvention with the pharmaceutically acceptable carrier. For example, thepharmaceutical composition can be used in the form of a parenteralinjection of an aseptic solution or suspension with water or any otherpharmaceutically acceptable solution. For example, the pharmaceuticalcomposition may be formulated with the antigen-binding molecule mixed ina unit dosage form required for generally accepted pharmaceuticalpractice, in appropriate combination with pharmacologically acceptablecarriers or media, specifically, sterilized water, physiological saline,plant oil, an emulsifier, a suspending agent, a surfactant, astabilizer, a flavoring agent, an excipient, a vehicle, a preservative,a binder, etc. Specific examples of the carrier can include lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate,polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, saccharide,carboxymethylcellulose, cornstarch, and inorganic salts. The amount ofthe active ingredient in such a preparation is determined such that anappropriate dose within the prescribed range can be achieved.

An aseptic composition for injection can be formulated according toconventional pharmaceutical practice using a vehicle such as injectabledistilled water. Examples of aqueous solutions for injection includephysiological saline, isotonic solutions containing glucose and otheradjuvants, for example, D-sorbitol, D-mannose, D-mannitol, and sodiumchloride. These solutions may be used in combination with an appropriatesolubilizer, for example, an alcohol (specifically, ethanol) or apolyalcohol (e.g., propylene glycol and polyethylene glycol), or anonionic surfactant, for example, polysorbate 80™ or HCO-50.

Examples of oily solutions include sesame oil and soybean oil. Thesesolutions may be used in combination with benzyl benzoate or benzylalcohol as a solubilizer. The solutions may be further mixed with abuffer (e.g., a phosphate buffer solution and a sodium acetate buffersolution), a soothing agent (e.g., procaine hydrochloride), a stabilizer(e.g., benzyl alcohol and phenol), and an antioxidant. The injectionsolutions thus prepared are usually charged into appropriate ampules.The pharmaceutical composition of the present invention is preferablyadministered parenterally. Specific examples of its dosage forms includeinjections, intranasal administration agents, transpulmonaryadministration agents, and percutaneous administration agents. Examplesof the injections include intravenous injection, intramuscularinjection, intraperitoneal injection, and subcutaneous injection,through which the pharmaceutical composition can be administeredsystemically or locally.

The administration method can be appropriately selected depending on theage and symptoms of a patient. The dose of a pharmaceutical compositioncontaining a polypeptide or a polynucleotide encoding the polypeptidecan be selected within a range of, for example, 0.0001 to 1000 mg/kg ofbody weight per dose. Alternatively, the dose can be selected within arange of, for example, 0.001 to 100000 mg/body of a patient, though thedose is not necessarily limited to these numeric values. Although thedose and the administration method vary depending on the weight, age,symptoms, etc. of a patient, those skilled in the art can appropriatelyselect the dose and the method.

The present invention also provides a method for treating cancer,comprising the step of administering the antigen-binding molecule of thepresent invention, the antigen-binding molecule of the present inventionfor use in the treatment of cancer, use of the antigen-binding moleculeof the present invention in the production of a therapeutic agent forcancer, and a process for producing a therapeutic agent for cancer,comprising the step of using the antigen-binding molecule of the presentinvention.

The three-letter codes and corresponding one-letter codes of amino acidsused herein are defined as follows: alanine: Ala and A, arginine: Argand R, asparagine: Asn and N, aspartic acid: Asp and D, cysteine: Cysand C, glutamine: Gln and Q, glutamic acid: Glu and E, glycine: Gly andG, histidine: His and H, isoleucine: Ile and I, leucine: Leu and L,lysine: Lys and K, methionine: Met and M, phenylalanine: Phe and F,proline: Pro and P, serine: Ser and S, threonine: Thr and T, tryptophan:Trp and W, tyrosine: Tyr and Y, and valine: Val and V.

Those skilled in the art should understand that one of or anycombination of two or more of the aspects described herein is alsoincluded in the present invention unless a technical contradictionarises on the basis of the technical common sense of those skilled inthe art.

All references cited herein are incorporated herein by reference intheir entirety.

The present invention will be further illustrated with reference toExamples below. However, the present invention is not intended to belimited by Examples below.

EXAMPLES [Example 1] Concept of Altered Immunoglobulin Variable (Fab)Region that Binds CD3 (First Antigen) and Another Antigen (SecondAntigen), but does not Bind to CD3 (First Antigen) and Another Antigen(Second Antigen) on Different Cells at Same Time

The binding of an immunoglobulin to two or more molecules of activatingFcγR at the same time or to activating FcγR and another antigen at thesame time causes the cross-linking reaction of the activating FcγR,which may in turn transduces FcγR ITAM signals, resulting in thepossible activation of immunocytes. One molecule of an IgG-type antibodyis capable of binding to only one FcγR molecule, as described above.Therefore, two or more molecules of activating FcγR are cross-linkedonly in the presence of an antigen to activate immunocytes.

When an IgG-type antibody binds to an antigen through its variableregion (Fab), this antibody is also capable of binding to one moleculeof FcγR through its Fc region at the same time therewith. This causesthe cross-linking between a cell expressing the antigen and a cellexpressing FcγR. Depending on the cell expressing the antigen, suchcross-linking between the antigen and FcγR may not be favorable.Specifically, when the antigen is, for example, CD3, a T cellcross-linked with an FcγR-expressing cell may cause immune activationsuch as cytokine release (J. Immunol. (1999) Aug. 1, 163 (3), 1246-52).In such a case, the Fc region can lose its binding activity against FcγRby the introduction of alteration to prevent the cross-linking reactionbetween the antigen and FcγR (Advanced Drug Delivery Reviews (2006) 58,640-656). Likewise, when antigens for IgG-type antibodies are, forexample, TNFR superfamily molecules (e.g., CD40, OX40, and CD27) or CD3and TLR (e.g., TLR2, TLR4, TLR8, and TLR9), the FcγR-mediatedcross-linking causes systemic immune activation. Thus, the binding atthe same time to these molecules expressed on separate cells is notfavorable.

Meanwhile, a conventional multispecific antibody binds to a plurality ofantigens at the same time. Depending on the combination of the antigens,the binding to a plurality of antigens at the same time may not befavorable. For example, integrin αvβ3, known as an adhesion molecule, isexpressed in many cancer cells and peritumoral blood vessels and assuch, is useful as a target molecule in tumor targeting (R. Haubner,PLoS Med., 2, e70 (2005)), whereas this molecule is also known to beexpressed in various normal cells (Thromb Haemost. 1998 November; 80(5): 726-34). Thus, the binding of the multispecific antibody to bothCD3 and integrin αvβ3 at the same time might damage normal cells due topotent cytotoxic activity mediated by T cells.

Accordingly, a possible method for controlling such unfavorablecross-linking reaction was dual binding Fab, which is one variable (Fab)region that binds to the first antigen through a portion thereof andbinds to the second antigen through a different portion that does notparticipate in this binding to the first antigen (FIG. 1). Provided thattwo proximally positioned moieties in one variable (Fab) region areessential for the binding to their respective antigens, as shown in FIG.1, the binding to the first antigen inhibits the binding to the secondantigen while the binding to the second antigen also inhibits thebinding to the first antigen. Thus, a modified antibody having theproperties of such dual binding Fab cannot bind to the first antigen andthe second antigen at the same time and therefore, presumably causes nocross-linking reaction between the first antigen and the second antigen(FIG. 2). Also, the dual binding Fab is considered to be capable ofbinding to both the first antigen and the second antigen at the sametime when the first antigen and the second antigen are not expressed oncell membranes, as with soluble proteins, or both reside on the samecell, but to neither bind to these antigens each expressed on adifferent cell at the same time nor cross-link these two cells (FIG. 3).On the other hand, an antigen (third antigen) binding to anothervariable (Fab) region may undergo cross-linking reaction with the firstantigen (FIG. 4) or may undergo cross-linking reaction with the secondantigen (FIG. 5). For this antibody, an Fc region binding to FcγR may beused as a constant region, or an Fc region having reduced bindingactivity against FcγR may be used as a constant region.

By use of the properties of such dual binding Fab, for example, atechnique of damaging cancer cells expressing a cancer antigen by theantibody-mediated redirection of T cells can be further provided with afunction of targeting integrin in cancer tissues and thereby achievehigher cancer specificity.

Briefly, if a variable (Fab) region can be modified as dual binding Fabto confer the following properties, an antibody having the effects asshown in FIG. 1 can be developed:

1. having binding activity against the first antigen;

2. having binding activity against the second antigen; and

3. not binding to the first antigen and the second antigen at the sametime.

The phrase “not bind to the first antigen and the second antigen at thesame time” also includes not cross-linking a cell expressing the firstantigen to a cell expressing the second antigen, or not binding to thefirst antigen and the second antigen each expressed on a different cell,at the same time. This phrase further includes the case where thevariable region is capable of binding to both the first antigen and thesecond antigen at the same time when the first antigen and the secondantigen are not expressed on cell membranes, as with soluble proteins,or both reside on the same cell, but cannot bind to the first antigenand the second antigen each expressed on a different cell, at the sametime.

Likewise, if a variable (Fab) region can be modified as dual binding Fabto confer the following properties, an antibody having, for example, theeffects as shown in FIG. 6 can be developed:

1. having binding activity against the first antigen on a T cell;

2. having binding activity against the second antigen on anantigen-presenting cell; and

3. not binding to the first antigen and the second antigen at the sametime.

[Example 2] Preparation of Anti-Human and Anti-Cynomolgus Monkey CD3εAntibody CE115

(2-1) Preparation of Hybridoma Using Rat Immunized with Cell ExpressingHuman CD3 and Cell Expressing Cynomolgus Monkey CD3

Each SD rat (female, 6 weeks old at the start of immunization, CharlesRiver Laboratories Japan, Inc.) was immunized with Ba/F3 cellsexpressing human CD3εγ or cynomolgus monkey CD3εγ as follows: at day 0(the priming date was defined as day 0), 5×10⁷ Ba/F3 cells expressinghuman CD3εγ were intraperitoneally administered together with a Freundcomplete adjuvant (Difco Laboratories, Inc.) to the rat. At day 14,5×10⁷ Ba/F3 cells expressing cynomolgus monkey CD3εγ wereintraperitoneally administered thereto together with a Freund incompleteadjuvant (Difco Laboratories, Inc.). Then, 5×10⁷ Ba/F3 cells expressinghuman CD3εγ and Ba/F3 cells expressing cynomolgus monkey CD3εγ wereintraperitoneally administered thereto a total of four times every otherweek in an alternate manner. One week after (at day 49) the finaladministration of CD3εγ, Ba/F3 cells expressing human CD3εγ wereintravenously administered thereto as a booster. Three days thereafter,the spleen cells of the rat were fused with mouse myeloma cells SP2/0according to a routine method using PEG1500 (Roche Diagnostics K.K.).Fusion cells, i.e., hybridomas, were cultured in an RPMI1640 mediumcontaining 10% FBS (hereinafter, referred to as 10% FBS/RPMI1640).

On the day after the fusion, (1) the fusion cells were suspended in asemifluid medium (Stemcell Technologies, Inc.). The hybridomas wereselectively cultured and also colonized.

Nine or ten days after the fusion, hybridoma colonies were picked up andinoculated at 1 colony/well to a 96-well plate containing a HATselective medium (10% FBS/RPMI1640, 2 vol % HAT 50× concentrate(Sumitomo Dainippon Pharma Co., Ltd.), and 5 vol % BM-Condimed H1 (RocheDiagnostics K.K.)). After 3- to 4-day culture, the culture supernatantin each well was recovered, and the rat IgG concentration in the culturesupernatant was measured. The culture supernatant confirmed to containrat IgG was screened for a clone producing an antibody specificallybinding to human CD3εγ by cell-ELISA using attached Ba/F3 cellsexpressing human CD3εγ or attached Ba/F3 cells expressing no human CD3εγ(FIG. 7). The clone was also evaluated for cross reactivity with monkeyCD3εγ by cell-ELISA using attached Ba/F3 cells expressing cynomolgusmonkey CD3εγ (FIG. 7).

(2-2) Preparation of Anti-Human and Anti-Monkey CD3ε Chimeric Antibody

Total RNA was extracted from each hybridoma cell using RNeasy Mini Kits(Qiagen N.V.), and cDNA was synthesized using SMART RACE cDNAAmplification Kit (BD Biosciences). The prepared cDNA was used in PCR toinsert the antibody variable region gene to a cloning vector. Thenucleotide sequence of each DNA fragment was determined using BigDyeTerminator Cycle Sequencing Kit (Applied Biosystems, Inc.) and a DNAsequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems, Inc.)according to the method described in the instruction manual includedtherein. CDRs and FRs of the CE115 H chain variable domain (SEQ ID NO:13) and the CE115 L chain variable domain (SEQ ID NO: 14) weredetermined according to the Kabat numbering.

A gene encoding a chimeric antibody H chain containing the rat antibodyH chain variable domain linked to a human antibody IgG1 chain constantdomain, and a gene encoding a chimeric antibody L chain containing therat antibody L chain variable domain linked to a human antibody kappachain constant domain were integrated to expression vectors for animalcells. The prepared expression vectors were used for the expression andpurification of the CE115 chimeric antibody (Reference Example 1).

(2-3) Preparation of EGFR_ERY22_CE115

Next, IgG against a cancer antigen (EGFR) was used as a backbone toprepare a molecule in a form with one Fab replaced with CD3ε-bindingdomains. In this operation, silent Fc having attenuated binding activityagainst FcgR (Fcγ receptor) was used, as in the case mentioned above, asFc of the backbone IgG. Cetuximab-VH (SEQ ID NO: 15) and Cetuximab-VL(SEQ ID NO: 16) constituting the variable region of cetuximab were usedas EGFR-binding domains. G1d derived from IgG1 by the deletion ofC-terminal Gly and Lys, A5 derived from G1d by the introduction of D356Kand H435R mutations, and B3 derived from G1d by the introduction of aK439E mutation were used as antibody H chain constant domains and eachcombined with Cetuximab-VH to prepare Cetuximab-VH-G1d (SEQ ID NO: 17),Cetuximab-VH-A5 (SEQ ID NO: 18), and Cetuximab-VH-B3 (SEQ ID NO: 19)according to the method of Reference Example 1. When the antibody Hchain constant domain was designated as H1, the sequence correspondingto the antibody H chain having Cetuximab-VH as a variable domain wasrepresented by Cetuximab-VH-H1.

In this context, the alteration of an amino acid is represented by, forexample, D356K. The first alphabet (which corresponds to D in D356K)means an alphabet that represents the one-letter code of the amino acidresidue before the alteration. The number (which corresponds to 356 inD356K) following the alphabet means the EU numbering position of thisaltered residue. The last alphabet (which corresponds to K in D356K)means an alphabet that represents the one-letter code of an amino acidresidue after the alteration.

EGFR_ERY22_CE115 (FIG. 8) was prepared by the exchange between the VHdomain and the VL domain of Fab against EGFR. Specifically, a series ofexpression vectors having an insert of each polynucleotide encodingEGFR_ERY22_Hk (SEQ ID NO: 20), EGFR_ERY22_L (SEQ ID NO: 21),CE115_ERY22_Hh (SEQ ID NO: 22), or CE115_ERY22_L (SEQ ID NO: 23) wasprepared by a method generally known to those skilled in the art, suchas PCR, using primers with an appropriate sequence added in the same wayas the aforementioned method.

The expression vectors were transferred in the following combination toFreeStyle 293-F cells where each molecule of interest was transientlyexpressed:

Molecule of Interest: EGFR_ERY22_CE115

Polypeptides encoded by the polynucleotides inserted in the expressionvectors: EGFR ERY22_Hk, EGFR ERY22_L, CE115_ERY22_Hh, and CE115_ERY22_L

(2-4) Purification of EGFR_ERY22_CE115

The obtained culture supernatant was added to Anti FLAG M2 column(Sigma-Aldrich Corp.), and the column was washed, followed by elutionwith 0.1 mg/mL FLAG peptide (Sigma-Aldrich Corp.). The fractioncontaining the molecule of interest was added to HisTrap HP column (GEHealthcare Japan Corp.), and the column was washed, followed by elutionwith the concentration gradient of imidazole. The fraction containingthe molecule of interest was concentrated by ultrafiltration. Then, thisfraction was added to Superdex 200 column (GE Healthcare Japan Corp.).Only a monomer fraction was recovered from the eluate to obtain eachpurified molecule of interest.

(2-5) Measurement of Cytotoxic Activity Using Human Peripheral BloodMononuclear Cell

(2-5-1) Preparation of Human Peripheral Blood Mononuclear Cell (PBMC)Solution

50 mL of peripheral blood was collected from each healthy volunteer(adult) using a syringe pre-filled with 100 μL of 1,000 units/mL of aheparin solution (Novo-Heparin 5,000 units for Injection, Novo NordiskA/S). The peripheral blood was diluted 2-fold with PBS(−) and thendivided into four equal parts, which were then added to Leucoseplymphocyte separation tubes (Cat. No. 227290, Greiner Bio-One GmbH)pre-filled with 15 mL of Ficoll-Paque PLUS and centrifuged in advance.After centrifugation (2,150 rpm, 10 minutes, room temperature) of theseparation tubes, a mononuclear cell fraction layer was separated. Thecells in the mononuclear cell fraction were washed once with Dulbecco'sModified Eagle's Medium containing 10% FBS (Sigma-Aldrich Corp.;hereinafter, referred to as 10% FBS/D-MEM). Then, the cells wereadjusted to a cell density of 4×10⁶ cells/mL with 10% FBS/D-MEM. Thecell solution thus prepared was used as a human PBMC solution in thesubsequent test.

(2-5-2) Measurement of Cytotoxic Activity

The cytotoxic activity was evaluated on the basis of the rate of cellgrowth inhibition using xCELLigence real-time cell analyzer (RocheDiagnostics). The target cells used were an SK-pca13a cell lineestablished by forcing an SK-HEP-1 cell line to express human EGFR.SK-pca13a was dissociated from the dish and inoculated at 100 μL/well(1×10⁴ cells/well) to an E-Plate 96 plate (Roche Diagnostics) to startthe assay of live cells using the xCELLigence real-time cell analyzer.On the next day, the plate was taken out of the xCELLigence real-timecell analyzer, and 50 μL of each antibody adjusted to each concentration(0.004, 0.04, 0.4, and 4 nM) was added to the plate. After reaction atroom temperature for 15 minutes, 50 μL (2×10⁵ cells/well) of the humanPBMC solution prepared in the preceding paragraph (2-5-1) was addedthereto. This plate was reloaded to the xCELLigence real-time cellanalyzer to start the assay of live cells. The reaction was carried outunder conditions of 5% CO₂ and 37° C. 72 hours after the addition ofhuman PBMC. The rate of cell growth inhibition (%) was determined fromthe cell index value according to the expression given below. A numericvalue after normalization against the cell index value immediatelybefore the addition of the antibody defined as 1 was used as the cellindex value in this calculation.

Rate of cell growth inhibition (%)=(A−B)×100/(A−1), wherein

A represents the average cell index value of wells non-supplemented withthe antibody (only the target cells and human PBMC), and B representsthe average cell index value of the wells supplemented with eachantibody. The test was conducted in triplicate.

The cytotoxic activity of EGFR_ERY22_CE115 containing CE115 was measuredwith PBMC prepared from human blood as effector cells. As a result, verystrong activity was confirmed (FIG. 9).

[Example 3] Preparation of Antibody that Binds to CD3 and Human Integrinαvβ3, but does not Bind to these Antigens at Same Time

As shown in FIGS. 1 to 6, the dual binding Fab is a molecule that bindsto CD3 (first antigen) and the antigen of interest (second antigen)through its variable (Fab) region, but does not bind to CD3 (firstantigen) and the antigen of interest (second antigen) at the same time.In the case of introducing amino acid alteration for binding to thesecond antigen to a CD3 (first antigen)-binding antibody Fab region, theamino acid alteration is usually introduced to both of two H chains or Lchains. As a result of introducing the alteration to both of the Hchains or the L chains, the two antibody Fabs become capable of bindingto two antigens, respectively. Thus, these two Fabs might bind to CD3(first antigen) and the antigen of interest (second antigen) at the sametime to cross-link them. Thus, one Fab of the antibody is prepared asFab binding to a third antigen or nothing, and the other Fab is preparedas dual binding Fab to prevent the cross-linking reaction between CD3(first antigen) and the antigen of interest (second antigen).

(3-1) Preparation of Antibody that Binds to CD3 and Human Integrin αvβ3,but does not Bind to these Antigens at Same Time

Integrin αvβ3, known as an adhesion molecule, is expressed in manycancer cells and peritumoral blood vessels and as such, is useful as atarget molecule in tumor targeting, whereas this molecule is also knownto be expressed in various normal cells (Thromb Haemost. 1998 November;80 (5): 726-34). Thus, binding to CD3 and integrin αvβ3 at the same timemight damage normal cells due to potent cytotoxic activity mediated by Tcells. Accordingly, it was assumed that an anti-EGFR antibody moleculecan target tumor cells expressing integrin αvβ3 without damaging normalcells, if a molecule that does not bind to CD3 and integrin αvβ3 at thesame time can be prepared. Thus, a study was conducted to obtain a dualbinding Fab molecule capable of binding to EGFR through one variableregion (Fab) and binding to the first antigen CD3 and the second antigenintegrin αvβ3 through the other variable region, but not capable ofbinding to CD3 and integrin αvβ3 at the same time.

Given that a “molecule that binds to CD3 through one Fab region underintegrin αvβ3-free conditions and binds to integrin αvβ3 through theother Fab region under CD3-free conditions” can be shown to be a“molecule that does not bind to integrin αvβ3 in a state bound with CD3or does not bind to CD3 in a state bound with integrin αvβ3”, it can beconcluded that a dual binding Fab molecule having the properties of dualbinding Fab of interest (i.e., the properties of being capable ofbinding to CD3 and the second antigen, but not binding to CD3 and thesecond antigen at the same time) has been developed successfully.

(3-2) Obtainment of Antibody Having Fab Region Binding to Integrin αvβ3

Possible methods for obtaining the dual binding Fab molecule were amethod using libraries and a method using the insertion of a peptideknown to have binding activity against a protein. An RGD (Arg-Gly-Asp)peptide is known as a peptide having binding activity against integrinαvβ3. Thus, the RGD peptide was inserted to the heavy chain loop of theCD3ε-binding antibody CE115 (heavy chain variable domain: SEQ ID NO: 13,light chain variable domain: SEQ ID NO: 14) to prepare eachheterodimerized antibody having EGFR-binding domains in one Fab and aCD3-binding domain and an integrin αvβ3-binding domain in the other Fabaccording to Reference Example 1. Specifically, a series of expressionvectors was prepared so as to have an insert of each polynucleotideencoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), orCE115_ERY22_L (SEQ ID NO: 23) as well as a polynucleotide encoding anyof the following fragments:

CE115_2 ERY22_Hh (SEQ ID NO: 24 with Kabat numbering positions 52b and53 substituted by K and N, respectively),

CE115_4 ERY22_Hh (SEQ ID NO: 25 with Kabat numbering positions 52b and54 substituted by S and N, respectively),

CE115_9 ERY22_Hh (SEQ ID NO: 26 with RGD inserted between Kabatnumbering positions 52a and 52b),

CE115_10 ERY22_Hh (SEQ ID NO: 27 with RGD inserted between Kabatnumbering positions 52b and 52c),

CE115_2 ERY22_Hh (SEQ ID NO: 28 with RGD inserted between Kabatnumbering positions 72 and 73),

CE115_17 ERY22_Hh (SEQ ID NO: 29 with Kabat numbering positions 52b and52c substituted by K and S, respectively),

CE115_47 ERY22_Hh (SEQ ID NO: 30 with RGD inserted between Kabatnumbering positions 98 and 99),

CE115_48 ERY22_Hh (SEQ ID NO: 31 with RGD inserted between Kabatnumbering positions 99 and 100), and

CE115_49 ERY22_Hh (SEQ ID NO: 32 with RGD inserted between Kabatnumbering positions 100 and 100a).

Also, an antibody (EH240-Kn125/EH240-H1076/L73; SEQ ID NO: 33/34/35)with the RGD (Arg-Gly-Asp) peptide inserted in an antibody CH3 regionreported in J. Biotech, 155, 193-202, 2011 was prepared as a controlaccording to Reference Example 1. This molecule binding to integrin αvβ3through its CH3 region is presumably capable of binding to CD3 andintegrin αvβ3 at the same time.

(3-3) Confirmation of Binding of Antibody to Integrin αvβ3

Each molecule with the RGD (Arg-Gly-Asp) peptide inserted in the Fabregion was evaluated for its binding to integrin αvβ3 by theelectrochemiluminescence method (ECL method). Specifically, biotin-antihuman IgG Ab (Southern Biotech) diluted with a TBS solution containing0.1% BSA, 0.1 g/L calcium chloride, and 0.1 g/L magnesium chloride(referred to as a dilution(+) solution), each antibody solution adjustedto 5 μg/mL or 1 μg/mL, and integrin αvβ3 (R&D Systems, Inc.) tagged withsulfo-tag were each added at 25 μL/well to Nunc-Immuno™ MicroWell™ 96well round plates (Nunc), and mixed, and the plate was then incubatedovernight at 4° C. to form an antibody-antigen complex. A TBS solutioncontaining 0.5% BSA, 0.1 g/L calcium chloride, and 0.1 g/L magnesiumchloride (referred to as a blocking(+) solution) was added at 150μL/well to streptavidin plate (MSD K.K.), and the plate was incubatedovernight at 4° C. After removal of the blocking solution, each well waswashed three times with 250 μL of a TBS solution containing 0.1 g/Lcalcium chloride and 0.1 g/L magnesium chloride (referred to as a TBS(+)solution). The antibody-antigen complex solution was added thereto at 75μL/well, and the plate was incubated at room temperature for 2 hours sothat the biotin-anti human IgG Ab bound to the streptavidin plate. Afterremoval of the antibody-antigen complex solution, each well was washedthree times with a TBS(+) solution, and READ buffer (MSD K.K.) was addedthereto at 150 μL/well, followed by the detection of the luminescencesignal of the sulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 11. The parent antibody EGFR ERY22_Hk/EGFRERY22_L/CE115_ERY22_Hh/CE115_ERY22_L exhibited no binding activityagainst integrin αvβ3, whereas all of EGFR ERY22_Hk/EGFR ERY22_L/CE115_2ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_4ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_9ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_10ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_2ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_17ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_47ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_48ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFR ERY22_L/CE115_49ERY22_Hh/CE115_ERY22_L were observed to bind to integrin αvβ3.

(3-4) Confirmation of Binding of Antibody to CD3 (CD3ε)

Next, each antibody having an integrin αvβ3-binding Fab region preparedin the previous section was evaluated for whether to retain bindingactivity against CD3 by the ECL method. Specifically, biotin-anti humanIgG Ab (Southern Biotech) diluted with a TBS solution containing 0.1%BSA (referred to as a dilution(−) solution), each antibody solutionadjusted to 5 μg/mL or 1 μg/mL, and CD3ε homodimer protein tagged withsulfo-tag were each added at 25 μL/well to Nunc-Immuno™ MicroWell™ 96well round plates (Nunc), and mixed, and the plate was then incubatedovernight at 4° C. to form an antibody-antigen complex. A TBS solutioncontaining 0.5% BSA (referred to as a blocking(−) solution) was added at150 μL/well to streptavidin plate (MSD K.K.), and the plate wasincubated overnight at 4° C. After removal of the blocking solution,each well was washed three times with 250 μL of a TBS(−) solution. Theantibody-antigen complex solution was added thereto at 75 μL/well, andthe plate was incubated at room temperature for 2 hours so that thebiotin-anti human IgG Ab bound to the streptavidin plate. After removalof the antibody-antigen complex solution, each well was washed threetimes with a TBS(−) solution, and READ buffer (MSD K.K.) was addedthereto at 150 μL/well, followed by the detection of the luminescencesignal of the sulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 12. All of the parent antibody EGFRERY22_Hk/EGFR ERY22_L/CE115_ERY22_Hh/CE115_ERY22_L as well as EGFRERY22_Hk/EGFR ERY22_L/CE115_2 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_4 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_9 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_10 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_2 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_17 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_47 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_48 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_49 ERY22_Hh/CE115_ERY22_L were observed to bind to CD3.

(3-5) Confirmation that Fab Region does not Bind to Integrin αvβ3 andCD3 at Same Time by ECL Method

As is evident from the results of the above paragraphs, the obtainedmolecules had binding activity against integrin αvβ3 and had bindingactivity against CD3. Next, each Fab region prepared in the aboveparagraphs was evaluated for whether to bind to CD3 (CD3ε) and integrinαvβ3 at the same time.

When a molecule with the RGD (Arg-Gly-Asp) peptide inserted in the Fabregion binds to integrin αvβ3 and CD3 at the same time, its binding toboth the antigens can be detected by the ECL method by adding integrinαvβ3 and biotinylated CD3 to the antibody solution. Specifically,biotinylated human CD3ε homodimer protein diluted with a dilution(+)solution, each antibody solution adjusted to 10 μg/mL or 5 μg/mL, andintegrin αvβ3 (R&D Systems, Inc.) tagged with sulfo-tag were each addedat 25 μL/well to Nunc-Immuno™ MicroWell™ 96 well round plates (Nunc),and mixed, and the plate was then incubated overnight at 4° C. to forman antibody-antigen complex. A blocking(+) solution was added at 150μL/well to streptavidin plate (MSD K.K.), and the plate was incubatedovernight at 4° C. After removal of the blocking solution, each well waswashed three times with 250 μL of a TBS(+) solution containing 0.1 g/Lcalcium chloride and 0.1 g/L magnesium chloride. The antibody-antigencomplex solution was added thereto at 75 μL/well, and the plate wasincubated at room temperature for 2 hours so that the biotin-anti humanIgG Ab bound to the streptavidin plate. After removal of theantibody-antigen complex solution, each well was washed three times witha TBS(+) solution, and READ buffer (MSD K.K.) was added thereto at 150μL/well, followed by the detection of the luminescence signal of thesulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIGS. 13 and 14. EGFR ERY22_Hk/EGFRERY22_L/CE115_2 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_2 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_17 ERY22_Hh/CE115_ERY22_L with the RGD (Arg-Gly-Asp)peptide inserted in the Fab region bound to integrin αvβ3 and CD3 at thesame time, resulting in the strong signal detected in the ECL assay. Bycontrast, EGFR ERY22_Hk/EGFR ERY22_L/CE115_9 ERY22_Hh/CE115_ERY22_L andEGFR ERY22_Hk/EGFR ERY22_L/CE115_48 ERY22_Hh/CE115_ERY22_L produced onlya weak signal in this assay (FIG. 13). All of EGFR ERY22_Hk/EGFRERY22_L/CE115_4 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_10 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_47 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_49 ERY22_Hh/CE115_ERY22_L rarely produced a detectablesignal in the ECL assay (FIG. 14). These results suggested that theseantibodies do not bind to integrin αvβ3 in a state bound with CD3.

(3-6) Discussion on Results of ECL Method Showing that Fab Region doesnot Bind to Integrin αvβ3 and CD3 at Same Time

As is evident from the results described above, the developed antibodyhad the properties of the dual binding Fab molecule binding to each ofCD3 (CD3ε) and integrin αvβ3 through one Fab, but not binding to CD3(CD3ε) and integrin αvβ3 at the same time. In this Example, the RGDpeptide binding to the second antigen integrin αvβ3 was inserted to thevariable region (Fab) of the antibody having this variable regionbinding to the first antigen CD3 to successfully obtain a molecule thatwas provided with the binding activity against the second antigen, butdid not bind to CD3 and the second antigen at the same time. By similarmethods, a peptide having binding activity against a protein asillustrated in WO2006036834 can be inserted to the Fab loop to obtain adual binding Fab molecule having binding activity against an arbitrarysecond antigen. The peptide exhibiting binding activity against aprotein can be obtained by preparing a peptide library by use of amethod generally known to those skilled in the art and selecting apeptide having the desired activity from the library (Pasqualini R.,Nature, 1996, 380 (6572): 364-6). Furthermore, a library ofantigen-binding molecules prepared by alteration to a larger length(extension) of loops in Fab as described in Example 5 may be used todevelop a dual binding Fab molecule having binding activity against anarbitrary second antigen. The variable regions against the first antigencan be obtained by various methods generally known to those skilled inthe art. Hence, it can be concluded that such libraries can be used todevelop dual binding Fab molecules that have binding activity against anarbitrary first antigen and an arbitrary second antigen, but cannot bindto the first antigen and the second antigen at the same time.

The results described above indicated that EGFR ERY22_Hk/EGFRERY22_L/CE115_4 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_10 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_47 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_49 ERY22_Hh/CE115_ERY22_L bind to CD3 and integrin αvβ3,but do not bind to CD3 and integrin αvβ3 at the same time. These resultsdemonstrated that EGFR ERY22_Hk/EGFR ERY22_L/CE115_4ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_10ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_47ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFR ERY22_L/CE115_49ERY22_Hh/CE115_ERY22_L are molecules having dual binding Fab, and suchmolecules can be developed.

[Example 4] Preparation of Antibody that Binds to CD3 and HumanToll-Like Receptor 2 (TLR2), but does not Bind to these Antigens at SameTime

(4-1) Preparation of Antibody that Binds to CD3 and Human TLR2, but doesnot Bind to these Antigens at Same Time

TLR2, known as a pattern recognition receptor, is expressed mainly onimmunocytes such as macrophages, dendritic cells, or B cells and isuseful as a target molecule activating the immunocytes. TLR2 is alsoknown to be expressed on normal cells other than immunocytes, such asepithelial cells or endothelial cells. The binding of a cancer antigenand CD3 at the same time recruits T cells expressing CD3 in a tumorenvironment so that the T cells damage the cancer cells. In this case,the binding of the cancer antigen and TLR2 at the same time can alsorecruit immunocytes expressing TLR2 in the tumor environment, presumablyactivating the immunocytes. The cancer cells damaged by the T cells aretaken up by the immunocytes recruited by TLR2. The antigen can beprocessed and presented on HLA to activate the T cells. This mightactivate T cells more strongly and also induce acquired immunity.However, binding to CD3 and TLR2 at the same time might damageimmunocytes and normal cells due to potent cytotoxic activity mediatedby T cells. Accordingly, it was assumed that immunocytes and normalcells expressing TLR2 can be recruited without damaging these cells, ifa molecule that does not bind to CD3 and TLR2 at the same time can beprepared. Thus, a study was conducted to obtain a dual binding Fabmolecule capable of binding to EGFR through one variable region (Fab)and binding to the first antigen CD3 and the second antigen TLR2 throughthe other variable region, but not capable of binding to CD3 and TLR2 atthe same time.

Given that a “molecule that binds to CD3 through one Fab region underTLR2-free conditions and binds to TLR2 through the other Fab regionunder CD3-free conditions” can be shown to be a “molecule that does notbind to TLR2 in a state bound with CD3 or does not bind to CD3 in astate bound with TLR2”, it can be concluded that a dual binding Fabmolecule having the properties of dual binding Fab of interest (i.e.,the properties of binding to CD3 and the second antigen, but not bindingto CD3 and the second antigen at the same time) has been developedsuccessfully.

(4-2) Obtainment of Antibody Having Fab Region Binding to TLR2

An RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known as a peptidehaving binding activity against human TLR2. Thus, the TRL2-bindingpeptide was inserted to the heavy chain loop of the CD3ε-bindingantibody CE115 (heavy chain variable domain: SEQ ID NO: 13, light chainvariable domain: SEQ ID NO: 14) to prepare each heterodimerized antibodyhaving EGFR-binding domains in one Fab and a CD3-binding domain and aTLR2-binding domain in the other Fab according to Reference Example 1.Specifically, a series of expression vectors was prepared so as to havean insert of each polynucleotide encoding EGFR ERY22_Hk (SEQ ID NO: 20),EGFR ERY22_L (SEQ ID NO: 21), or CE115_ERY22_L (SEQ ID NO: 23) as wellas a polynucleotide encoding any of the following fragments:

CE115_DU21 ERY22_Hh (SEQ ID NO: 37 with the TRL2-binding peptideinserted between Kabat numbering positions 52b and 52c),

CE115_DU22 ERY22_Hh (SEQ ID NO: 38 with the TRL2-binding peptideinserted between Kabat numbering positions 52b and 52c),

CE115_DU26 ERY22_Hh (SEQ ID NO: 39 with the TRL2-binding peptideinserted between Kabat numbering positions 72 and 73), and

CE115_DU27 ERY22_Hh (SEQ ID NO: 40 with the TRL2-binding peptideinserted between Kabat numbering positions 72 and 73).

Also, an antibody (CE115_ERY22_DU42_Hh, SEQ ID NO: 41) with theTLR2-binding peptide added to the C terminus of the CH3 region, and anantibody (CE115_ERY22_DU43_Hh, SEQ ID NO: 42) in which a TLR2-bindingpeptide having Cys residues at both ends was added to the C terminus ofthe CH3 region were prepared as controls according to ReferenceExample 1. These molecules binding to TLR2 through their CH3 regions arepresumably capable of binding to CD3 and TLR2 at the same time.

(4-3) Confirmation of Binding of Antibody to TLR2

Each molecule with the TLR2-binding peptide inserted in the Fab regionwas evaluated for its binding to TLR2 by the electrochemiluminescencemethod (ECL method). Specifically, biotin-anti human IgG Ab (SouthernBiotech) diluted with a TBS solution containing 0.1% BSA (referred to asa dilution(−) solution), each antibody solution adjusted to 5 μg/mL or 1μg/mL, and TLR2 (Abnova Corp.) tagged with sulfo-tag were each added at25 μL/well to Nunc-Immuno™ MicroWell™ 96 well round plates (Nunc), andmixed, and the plate was then incubated overnight at 4° C. to form anantibody-antigen complex. A TBS solution containing 0.5% BSA (referredto as a blocking(−) solution) was added at 150 μL/well to streptavidinplate (MSD K.K.), and the plate was incubated overnight at 4° C. Afterremoval of the blocking solution, each well was washed three times with250 μL of a TBS(−) solution. The antibody-antigen complex solution wasadded thereto at 75 μL/well, and the plate was incubated at roomtemperature for 2 hours so that the biotin-anti human IgG Ab bound tothe streptavidin plate. After removal of the antibody-antigen complexsolution, each well was washed three times with a TBS(−) solution, andREAD buffer (MSD K.K.) was added thereto at 150 μL/well, followed by thedetection of the luminescence signal of the sulfo-tag using SectorImager 2400 (MSD K.K.).

The results are shown in FIG. 15. The parent antibody EGFR ERY22_Hk/EGFRERY22_L/CE115_ERY22_Hh/CE115_ERY22_L exhibited no binding activityagainst TLR2, whereas all of EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU21ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU22ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU26ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU27ERY22_Hh/CE115_ERY22_L were observed to bind to TLR2.

(4-4) Confirmation of Binding of Antibody to CD3 (CD3ε)

Next, each antibody having a TLR2-binding Fab region prepared in theprevious section was evaluated for whether to retain binding activityagainst CD3 (CD3ε) by the ECL method. Specifically, biotin-anti humanIgG Ab (Southern Biotech) diluted with a TBS solution containing 0.1%BSA (referred to as a dilution(−) solution), each antibody solutionadjusted to 5 μg/mL or 1 μg/mL, and CD3ε homodimer protein tagged withsulfo-tag were each added at 25 μL/well to Nunc-Immuno™ MicroWell™ 96well round plates (Nunc), and mixed, and the plate was then incubatedovernight at 4° C. to form an antibody-antigen complex. A TBS solutioncontaining 0.5% BSA (referred to as a blocking(−) solution) was added at150 μL/well to streptavidin plate (MSD K.K.), and the plate wasincubated overnight at 4° C. After removal of the blocking solution,each well was washed three times with 250 μL of a TBS(−) solution. Theantibody-antigen complex solution was added thereto at 75 μL/well, andthe plate was incubated at room temperature for 2 hours so that thebiotin-anti human IgG Ab bound to the streptavidin plate. After removalof the antibody-antigen complex solution, each well was washed threetimes with a TBS(−) solution, and READ buffer (MSD K.K.) was addedthereto at 150 μL/well, followed by the detection of the luminescencesignal of the sulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 16. All of the parent antibody EGFRERY22_Hk/EGFR ERY22_L/CE115_ERY22_Hh/CE115_ERY22_L as well as EGFRERY22_Hk/EGFR ERY22_L/CE115_DU21 ERY22_Hh/CE115_ERY22_L, EGFRERY22_Hk/EGFR ERY22_L/CE115_DU22 ERY22_Hh/CE115_ERY22_L, EGFRERY22_Hk/EGFR ERY22_L/CE115_DU26 ERY22_Hh/CE115_ERY22_L, and EGFRERY22_Hk/EGFR ERY22_L/CE115_DU27 ERY22_Hh/CE115_ERY22_L were observed tobind to CD3.

(4-5) Confirmation that Fab Region does not Bind to TLR2 and CD3 at SameTime by ECL Method

As is evident from the results of the above paragraphs, the obtainedmolecules had binding activity against TLR2 and had binding activityagainst CD3. Next, each Fab region prepared in the above paragraphs wasevaluated for whether to bind to CD3 and TLR2 at the same time.

When a molecule with the TLR2-binding peptide inserted in the Fab regionbinds to TLR2 and CD3 at the same time, its binding to both the antigenscan be detected by the ECL method by adding TLR2 and biotinylated CD3 tothe antibody solution. Specifically, biotinylated human CD3ε homodimerprotein diluted with a dilution(−) solution, each antibody solutionadjusted to 10 μg/mL or 5 μg/mL, and TLR2 (R&D Systems, Inc.) taggedwith sulfo-tag were each added at 25 μL/well to Nunc-Immuno™ MicroWell™96 well round plates (Nunc), and mixed, and the plate was then incubatedovernight at 4° C. to form an antibody-antigen complex. A blocking(−)solution was added at 150 μL/well to streptavidin plate (MSD K.K.), andthe plate was incubated overnight at 4° C. After removal of the blockingsolution, each well was washed three times with 250 μL of a TBS(−)solution containing 0.1 g/L calcium chloride and 0.1 g/L magnesiumchloride. The antibody-antigen complex solution was added thereto at 75μL/well, and the plate was incubated at room temperature for 2 hours sothat the biotin-anti human IgG Ab bound to the streptavidin plate. Afterremoval of the antibody-antigen complex solution, each well was washedthree times with a TBS(−) solution, and READ buffer (MSD K.K.) was addedthereto at 150 μL/well, followed by the detection of the luminescencesignal of the sulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 17. EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU42ERY22_Hh/CE115_ERY22_L and EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU43ERY22_Hh/CE115_ERY22_L with the TLR2-binding peptide added to the CH3region bound to TLR2 and CD3 at the same time, resulting in the strongsignal detected in the ECL assay. By contrast, all of EGFR ERY22_Hk/EGFRERY22_L/CE115_DU21 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_DU22 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_DU26 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_DU27 ERY22_Hh/CE115_ERY22_L rarely produced a detectablesignal in the ECL assay. These results suggested that these antibodiesdo not bind to TLR2 in a state bound with CD3.

(4-6) Discussion on Results of ECL Method Showing that Fab Region doesnot Bind to TLR2 and CD3 at Same Time

As is evident from the results described above, the developed antibodyhad the properties of the dual binding Fab molecule binding to each ofCD3 and TLR2 through one Fab, but not binding to CD3 and TLR2 at thesame time. In this Example, the RWGYHLRDRKYKGVRSHKGVPR peptide bindingto the second antigen TLR2 was inserted to the variable region (Fab) ofthe antibody having this variable region binding to the first antigenCD3 to successfully obtain a molecule that was provided with the bindingactivity against the second antigen, but did not bind to CD3 and thesecond antigen at the same time. By similar methods, a peptide havingbinding activity against a protein as illustrated in WO2006036834 can beinserted to the Fab loop to obtain a dual binding Fab molecule havingbinding activity against an arbitrary second antigen. The peptideexhibiting binding activity against a protein can be obtained bypreparing a peptide library by use of a method generally known to thoseskilled in the art and selecting a peptide having the desired activityfrom the library (Pasqualini R., Nature, 1996, 380 (6572): 364-6).Furthermore, a library of antigen-binding molecules prepared byalteration to a larger length (extension) of loops in Fab as describedin Example 5 may be used to develop a dual binding Fab molecule havingbinding activity against an arbitrary second antigen. The variableregions against the first antigen can be obtained by various methodsgenerally known to those skilled in the art. Hence, it can be concludedthat such libraries can be used to develop dual binding Fab moleculesthat have binding activity against an arbitrary first antigen and anarbitrary second antigen, but cannot bind to the first antigen and thesecond antigen at the same time.

The results described above indicated that EGFR ERY22_Hk/EGFRERY22_L/CE115_DU21 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_DU22 ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFRERY22_L/CE115_DU26 ERY22_Hh/CE115_ERY22_L, and EGFR ERY22_Hk/EGFRERY22_L/CE115_DU27 ERY22_Hh/CE115_ERY22_L bind to CD3 and TLR2, but donot bind to CD3 and TLR2 at the same time. These results demonstratedthat EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU21 ERY22_Hh/CE115_ERY22_L, EGFRERY22_Hk/EGFR ERY22_L/CE115_DU22 ERY22_Hh/CE115_ERY22_L, EGFRERY22_Hk/EGFR ERY22_L/CE115_DU26 ERY22_Hh/CE115_ERY22_L, and EGFRERY22_Hk/EGFR ERY22_L/CE115_DU27 ERY22_Hh/CE115_ERY22_L are moleculeshaving dual binding Fab, and such molecules can be developed.

[Example 5] Antibody Alteration for Preparation of Antibody Binding toCD3 and Second Antigen

(5-1) Study on Insertion Site and Length of Peptide Capable of Bindingto Second Antigen

A study was conducted to obtain a dual binding Fab molecule capable ofbinding to a cancer antigen through one variable region (Fab) andbinding to the first antigen CD3 and the second antigen through theother variable region, but not capable of binding to CD3 and the secondantigen at the same time. A GGS peptide was inserted to the heavy chainloop of the CD3ε-binding antibody CE115 to prepare each heterodimerizedantibody having EGFR-binding domains in one Fab and CD3-binding domainsin the other Fab according to Reference Example 1.

Specifically, EGFR ERY22_Hk/EGFR ERY22_L/CE115_CE31ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/43/23) with GGS insertedbetween K52B and S52c in CDR2, EGFR ERY22_Hk/EGFR ERY22_L/CE115 CE32ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/44/23) with a GGSGGS peptide(SEQ ID NO: 90) inserted at this position, and EGFR ERY22_Hk/EGFRERY22_L/CE115_CE33 ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/45/23) witha GGSGGSGGS peptide (SEQ ID NO: 91) inserted at this position wereprepared. Likewise, EGFR ERY22_Hk/EGFR ERY22_L/CE115_CE34ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/46/23) with GGS insertedbetween D72 and D73 (loop) in FR3, EGFR ERY22_Hk/EGFR ERY22_L/CE115_CE35ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/47/23) with a GGSGGS peptide(SEQ ID NO: 90) inserted at this position, and EGFR ERY22_Hk/EGFRERY22_L/CE115_CE36 ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/48/23) witha GGSGGSGGS peptide (SEQ ID NO: 91) inserted at this position wereprepared. In addition, EGFR ERY22_Hk/EGFR ERY22_L/CE115 CE37ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/49/23) with GGS insertedbetween A99 and Y100 in CDR3, EGFR ERY22_Hk/EGFR ERY22_L/CE115 CE38ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/50/23) with a GGSGGS peptideinserted at this position, and EGFR ERY22_Hk/EGFR ERY22_L/CE115_CE39ERY22_Hh/CE115_ERY22_L ((SEQ ID NO: 20/21/51/23) with a GGSGGSGGSpeptide inserted at this position were prepared.

(5-2) Confirmation of Binding of GGS Peptide-Inserted CE115 Antibody toCD3ε

The binding activity of each prepared antibody against CD3ε wasconfirmed using Biacore T100. A biotinylated CD3ε epitope peptide wasimmobilized to a CM5 chip via streptavidin, and the prepared antibodywas injected thereto as an analyte and analyzed for its bindingaffinity.

The results are shown in Table 2. The binding affinity of CE35, CE36,CE37, CE38, and CE39 for CD3ε was equivalent to the parent antibodyCE115. This indicated that a peptide binding to the second antigen canbe inserted into their loops. The binding affinity was not reduced inGGSGGSGGS-inserted CE36 or CE39. This indicated that the insertion of apeptide up to at least 9 amino acids to these sites does not influencethe binding activity against CD3ε.

TABLE 2 Insertion Sample ka kd KD position Linker CE115_M 1.5E+059.8E−03 6.7E−08 CE31 2.3E+05 3.5E−02 1.5E−07 K52b-S52c GS3 CE32 8.5E+041.8E−02 2.1E−07 K52b-S52c GS6 CE33 4.9E+05 1.1E−01 2.3E−07 K52b-S52c GS9CE34 1.1E+05 1.3E−02 1.2E−07 D72-D73 GS3 CE35 1.3E+05 1.1E−02 8.7E−08D72-D73 GS6 CE36 1.2E+05 1.2E−02 9.9E−08 D72-D73 GS9 CE37 2.2E+052.0E−02 9.4E−08 A99-Y100 GS3 CE38 2.0E+05 1.7E−02 8.7E−08 A99-Y100 GS6CE39 1.6E+05 1.4E−02 9.1E−08 A99-Y100 GS9

These results indicated that the antibody capable of binding to CD3 andthe second antigen, but does not bind to these antigens at the same timecan be prepared by obtaining an antibody binding to the second antigenusing such peptide-inserted CE115.

In this context, a library can be prepared by altering at random theamino acid sequence of the peptide for use in insertion or substitutionaccording to a method known in the art such as site-directed mutagenesis(Kunkel et al., Proc. Natl. Acad. Sci. U.S.A. (1985) 82, 488-492) oroverlap extension PCR, and comparing the binding activity, etc., of eachaltered form according to the aforementioned method to determine aninsertion or substitution site that permits exertion of the activity ofinterest even after alteration of the amino acid sequence, and the typesand length of amino acids of this site.

[Example 6] Library Design for Obtaining Antibody Binding to CD3 andSecond Antigen

(6-1) Antibody Library for Obtaining Antibody Binding to CD3 and SecondAntigen (Also Referred to as Dual Fab Library)

In the case of selecting CD3 (CD3ε) as the first antigen, examples of amethod for obtaining an antibody binding to CD3 (CD3ε) and an arbitrarysecond antigen include the following 6 methods:

1. a method which involves inserting a peptide or a polypeptide bindingto the second antigen to a Fab domain binding to the first antigen (thismethod includes the peptide insertion shown in Example 3 or 4 as well asa G-CSF insertion method illustrated in Angew Chem Int Ed Engl. 2013Aug. 5; 52 (32): 8295-8), wherein the binding peptide or polypeptide maybe obtained from a peptide- or polypeptide-displaying library, or thewhole or a portion of a naturally occurring protein may be used;2. a method which involves preparing an antibody library such thatvarious amino acids appear positions that permit alteration to a largerlength (extension) of Fab loops as shown in Example 5, and obtaining Fabhaving binding activity against an arbitrary second antigen from theantibody library by using the binding activity against the antigen as anindex;3. a method which involves identifying amino acids that maintain bindingactivity against CD3 by use of an antibody prepared by site-directedmutagenesis from a Fab domain previously known to bind to CD3, andobtaining Fab having binding activity against an arbitrary secondantigen from an antibody library in which the identified amino acidsappear by using the binding activity against the antigen as an index;4. the method 3 which further involves preparing an antibody librarysuch that various amino acids appear positions that permit alteration toa larger length (extension) of Fab loops, and obtaining Fab havingbinding activity against an arbitrary second antigen from the antibodylibrary by using the binding activity against the antigen as an index;5. the method 1, 2, 3, or 4 which further involves altering theantibodies such that glycosylation sequences (e.g., NxS and NxT whereinx is an amino acid other than P) appear to add thereto sugar chains thatare recognized by sugar chain receptors (e.g., high-mannose-type sugarchains are added thereto and thereby recognized by high-mannosereceptors; it is known that the high-mannose-type sugar chains areobtained by the addition of kifunensine at the time of antibodyexpression (mAbs. 2012 July-August; 4 (4): 475-87)); and6. the method 1, 2, 3, or 4 which further involves adding theretodomains (polypeptides, sugar chains, and nucleic acids typified by TLRagonists) each binding to the second antigen through a covalent bond byinserting Cys, Lys, or a non-natural amino acid to loops or sites foundto be alterable to various amino acids or substituting these sites withCys, Lys, or a non-natural amino acid (this method is typified byantibody drug conjugates and is a method for conjugation to Cys, Lys, ora non-natural amino acid through a covalent bond (described in mAbs 6:1, 34-45; January/February 2014; WO2009/134891 A2; and Bioconjug Chem.2014 Feb. 19; 25 (2): 351-61)).

The dual binding Fab that binds to the first antigen and the secondantigen, but does not bind to these antigens at the same time isobtained by use of any of these methods, and can be combined withdomains (referred to as the other variable region, which is described inExample 1) binding to an arbitrary third antigen by a method generallyknown to those skilled in the art, for example, common L chains,CrossMab, or Fab arm exchange.

(6-2) Preparation of One-Amino Acid Alteration Antibody of CD3(CD3ε)-Binding Antibody Using Site-Directed Mutagenesis

A VH domain CE115HA000 (SEQ ID NO: 52) and a VL domain GLS3000 (SEQ IDNO: 53) were selected as template sequences for a CD3 (CD3ε)-bindingantibody. Each domain was subjected to amino acid alteration at a sitepresumed to participate in antigen binding according to ReferenceExample 1. Also, pE22Hh (sequence derived from natural IgG1 CH1 andsubsequent sequences by the alteration of L234A, L235A, N297A, D356C,T366S, L368A, and Y407V, the deletion of a C-terminal GK sequence, andthe addition of a DYKDDDDK sequence (SEQ ID NO: 89); SEQ ID NO: 54) wasused as an H chain constant domain, and a kappa chain (SEQ ID NO: 55)was used as an L chain constant domain. The alteration sites are shownin Table 3. For CD3 (CD3ε)-binding activity evaluation, each one-aminoacid alteration antibody was obtained as a one-arm antibody (naturallyoccurring IgG antibody lacking one of the Fab domains). Specifically, inthe case of H chain alteration, the altered H chain linked to theconstant domain pE22Hh, and KnO10G3 (naturally occurring IgG1 amino acidsequence from position 216 to the C terminus having C220S, Y349C, T366W,and H435R alterations; SEQ ID NO: 56) were used as H chains, and GLS3000linked at the 3′ side to the kappa chain was used as an L chain. In thecase of L chain alteration, the altered L chain linked at the 3′ side tothe kappa chain was used as an L chain, and CE115HA000 linked at the 3′side to pE22Hh, and Kn010G3 were used as H chains. These sequences wereexpressed and purified in FreeStyle 293 cells (which employed the methodof Reference Example 1).

TABLE 3 H chain alteration site Domain FR1 CDR1 FR2 CDR2 Kabat numbering11 16 19 28 29 30 31 32 33 35 43 50 51 52 52a 52b 52c 53 54 55 Aminoacid V R R T F S N A W H K Q I K A K S N N Y before substitution DomainCDR2 Kabat numbering 56 57 58 59 60 61 62 64 65 Amino acid A T Y Y A E SK G before substitution Domain FR3 CDR3 FR4 Kabat numbering 72 73 74 7576 77 78 82a 95 96 97 98 99 100 100a 100b 100c 101 102 105 Amino acid DD S K N S L N V H Y G A Y Y G V D A Q before substitution L chainalteration site Domain CDR1 FR2 Kabat numbering 24 25 26 27 27a 27b 27c27d 27e 28 29 30 31 32 33 34 45 Amino acid R S S Q S L V H S N R N T Y LH Q before substitution Domain CDR2 FR3 CDR3 FR4 Kabat numbering 50 5152 53 54 55 56 74 77 89 90 91 92 93 94 95 96 97 107 Amino K V S N R F SK R G Q G T Q V P Y T K acid before substitution

(6-3) Evaluation of Binding of One-Amino Acid Alteration Antibody to CD3

Each one-amino acid altered form constructed, expressed, and purified inthe paragraph (6-2) was evaluated using Biacore T200 (GE HealthcareJapan Corp.). An appropriate amount of CD3ε homodimer protein wasimmobilized onto Sensor chip CM4 (GE Healthcare Japan Corp.) by theamine coupling method. Then, the antibody having an appropriateconcentration was injected thereto as an analyte and allowed to interactwith the CD3ε homodimer protein on the sensor chip. Then, the sensorchip was regenerated by the injection of 10 mmol/L glycine-HCl (pH 1.5).The assay was conducted at 25° C., and HBS-EP+ (GE Healthcare JapanCorp.) was used as a running buffer. From the assay results, thedissociation constant K_(D) (M) was calculated using single-cyclekinetics model (1:1 binding RI=0) for the amount bound and thesensorgram obtained in the assay. Each parameter was calculated usingBiacore T200 Evaluation Software (GE Healthcare Japan Corp.).

(6-3-1) Alteration of H Chain

Table 4 shows the results of the ratio of the amount of each H chainaltered form bound to the amount of the corresponding unaltered antibodyCE115HA000 bound. Specifically, when the amount of the antibodycomprising CE115HA000 bound was defined as X and the amount of the Hchain one-amino acid altered form bound was defined as Y, a value of Z(ratio of amounts bound)=Y/X was used. As shown in FIG. 18, a very smallamount bound was observed in the sensorgram for Z of less than 0.8,suggesting the possibility that the dissociation constant K_(D) (M)cannot be calculated correctly. Table 5 shows the dissociation constantK_(D) (M) ratio of each H chain altered form to CE115HA000 (=KD value ofCE115HA000/KD value of the altered form).

When Z shown in Table 4 is 0.8 or more, the altered form is consideredto maintain the binding relative to the corresponding unaltered antibodyCE115HA000. Therefore, an antibody library designed such that theseamino acids appear can serve as a dual Fab library.

TABLE 4 Region FR1 CDR1 FR2 CDR2 Kabat numbering 11 16 19 28 29 30 31 3233 35 43 50 51 52 52a Original amino V R R T F S N A W H K Q I K A acid(wt) A 0.5 0.1 0.17 0.24 D 0.56 0.86 0.37 0.1 0.2 0.27 0.29 0.25 1.34 E0.88 0.19 0.9 0.26 0.55 0.26 0.57 F 0.62 0.65 0.21 0.17 G 1.01 0.39 0.220.81 H 0.68 0.13 0.22 I 0.81 0.12 0.4 0.33 K 1.01 0.15 0.33 L 1 0.1 0.110.23 M 0.29 N 0.35 0.17 0.34 P 0.15 Q 0.0 0.49 0.13 0.99 R 1.14 0.140.91 S 0.91 0.81 0.23 0.24 0.28 1.05 T 0.8 0.26 V 0.36 0.22 0.52 W 0.630.22 0.22 Y 0.64 0.33 0.66 0.16 0.25 0.18 0.31 Region CDR2 Kabatnumbering 52b 52c 53 54 55 56 57 58 59 60 61 62 64 65 Original amino K SN N Y A T Y Y A E S K G acid (wt) A 0.67 0.96 0.7 0.86 0.98 0.22 0.851.09 0.82 D 0.27 0.6 0.39 0.62 0.45 0.51 0.11 0.7 0.99 0.91 0.92 0.720.76 E 0.86 0.94 0.92 0.74 0.78 F 1.13 1.12 G 0.97 0.5 0.98 0.65 0.61 H0.76 I 0.68 0.61 K 1.19 0.78 1.2 1.35 1.32 0.3 1.19 L 0.61 0.98 0.94 0.80.27 M N 0.27 0.67 0.97 0.33 P 1.07 1 Q 0.6 1.04 1.1 0.84 0.76 0.19 1.070.89 R 1.11 S 0.68 0.83 0.84 0.26 0.18 0.94 0.84 T V 0.93 W 0.88 Y 0.741.11 0.63 1.03 0.66 Region FR3 CDR3 Kabat numbering 72 73 74 75 76 77 7882a 95 96 Original amino D D S K N S L N V H acid (wt) A 1.41 0.83 1.050.11 0.35 D 0.73 0.24 0.09 E 1.05 0.73 0.24 F G 1.07 0.19 0.43 H I 1.34K 0.87 0.64 0.35 L 0.14 M N 0.94 P 0.91 0.12 Q 0.42 R 1.04 1.01 0.46 S0.92 0.22 0.44 T 0.63 0.84 0.9 V 1.43 W Y 0.17 Region CDR3 FR4 Kabatnumbering 97 98 99 100 100a 100b 100c 101 102 105 Original amino Y G A YY G V D A Q acid (wt) A 0.16 1.1 0.9 0.62 1.26 D 0.24 0.26 0.28 0.520.31 0.27 0.44 E 0.26 0.46 0.94 F 1.43 0.87 0.3 0.75 G 0.18 1.07 1.231.38 H 1.58 1.21 I 1.18 1.48 K 2.83 1.48 1.07 0.9 0.63 L 1.13 0.7 0.480.27 0.62 M 1.2 N 2.02 P 0.11 1.02 0.48 0.2 0.2 0.14 Q 1.22 0.91 0.80.56 2.35 R 0.27 2.96 0.24 S 0.18 1.01 0.82 0.81 0.64 0.52 1.16 T 1.050.84 0.79 V 0.6 1.33 1.43 W 1.03 Y 2.22 1.59 0.23 0.49 0.91

TABLE 5 Region FR1 CDR1 FR2 CDR2 Kabat numbering 11 16 19 28 29 30 31 3233 35 43 50 51 52 52a Original amino V R R T F S N A W H K Q I K A acid(wt) A 0.96 29.99 25.04 22.83 D 0.93 0.79 1.14 1693.03 68.99 75.37 6.37166 1.35 E 0.74 70.35 0.88 16738.09 0.84 18.4 0.88 F 1.24 0.66 53.594.04 G 0.93 1.37 45.77 0.61 H 0.96 4.96 2.65 I 0.62 7.23 1.21 3.54 K0.97 14.45 0.71 L 0.83 56573.23 4.8 1.41 M 3.98 N 2.88 1.48 3.29 P 5 Q0.87 0.94 4.8 0.89 R 0.98 15429.77 0.8 S 0.79 0.67 2.93 47.38 92.1 0.82T 0.81 4.4 V 2.94 28.08 0.95 W 1.07 50.42 2.69 Y 1.1 2.11 0.69 119458.1349.09 6.47 7.71 Region CDR2 Kabat numbering 52b 52c 53 54 55 56 57 58 5960 61 62 64 65 Original amino K S N N Y A T Y Y A E S K G acid (wt) A0.58 0.67 0.55 0.58 0.87 1.06 0.74 0.94 0.81 D 0.56 0.55 0.55 0.59 0.890.71 4.81 0.66 0.94 0.9 0.87 0.76 0.61 E 0.61 0.88 0.82 0.84 0.61 F 0.930.97 G 0.81 0.95 0.84 0.99 0.59 H 0.55 I 0.57 0.81 K 0.88 0.79 0.82 1.321.22 0.66 0.99 L 0.61 0.94 0.91 0.77 1.21 M N 0.43 0.84 0.9 1.86 P 0.820.77 Q 0.62 0.97 1.05 0.8 0.74 1.24 0.85 0.87 R 0.91 S 0.58 0.59 0.575.65 1.22 0.79 0.85 T V 0.82 W 0.69 Y 0.61 0.87 0.94 1.03 0.63 RegionFR3 CDR3 Kabat numbering 72 73 74 75 76 77 78 82a 95 96 Original amino DD S K N S L N V H acid (wt) A 1.19 0.73 0.77 3.15 1 D 0.56 108.01 7.27 E0.73 0.56 50.46 F G 0.78 78256.33 0.8 H I 1.08 K 0.74 1.15 1.56 L 3.14 MN 0.7 P 0.7 87044.4 Q 1.36 R 0.79 0.88 1.59 S 0.84 4.61 1.15 T 0.78 0.750.83 V 1.17 W Y 6.67 Region CDR3 FR4 Kabat numbering 97 98 99 100 100a100b 100c 101 102 105 Original amino Y G A Y Y G V D A Q acid (wt) A41309 0.98 0.92 0.66 0.86 D 64.7 2.36 1.03 0.63 1.2 6.25 1.64 E 7.291.31 0.89 F 1.15 0.98 4.37 0.73 G 47212.8 0.97 1.01 3.16 H 1.14 0.91 I1.73 1.29 K 1.85 1.4 0.93 0.79 4.32 L 1 0.67 0.57 5.84 0.71 M 1.94 N2.28 P 12428.6 0.88 1.3 0.97 43.42 3.51 Q 1.04 0.85 0.77 0.51 3.55 R23179.6 4.69 5.55 S 1176 0.98 0.76 0.7 0.59 1.25 0.91 T 0.93 0.93 0.62 V0.92 1.18 1.27 W 0.96 Y 2.75 1.25 51.41 0.97 1

(6-3-2) Alteration of L Chain

Table 6 shows the results of the ratio of the amount of each L chainaltered form bound to the amount of the corresponding unaltered antibodyGLS3000 bound. Specifically, when the amount of the GLS3000-containingantibody bound was defined as X and the amount of the L chain one-aminoacid altered form bound was defined as Y, a value of Z (ratio of amountsbound)=Y/X was used. As shown in FIG. 18, a very small amount bound wasobserved in the sensorgram for Z of less than 0.8, suggesting thepossibility that the dissociation constant K_(D) (M) cannot becalculated correctly. Table 7 shows the dissociation constant K_(D) (M)ratio of each L chain altered form to GLS3000.

When Z shown in Table 6 is 0.8 or more, the altered form is consideredto maintain the binding relative to the corresponding unaltered antibodyGLS3000. Therefore, an antibody library designed such that these aminoacids appear can serve as a dual Fab library.

TABLE 6 Region CDR1 Kabat numbering 24 25 26 27 27a 27b 27c 27d 27eOriginal amino R S S Q S L V H S acid A 0.86 0.92 0.48 1.03 D 0.75 0.180.86 0.85 0.79 0.17 0.32 0.22 0.69 E 0.83 0.21 0.74 0.88 0.81 0.17 0.610.23 0.76 F 0.42 0.63 1.32 G 0.89 1.03 0.3 1.04 H 1.23 I 0.53 1 1.190.96 0.26 1.07 K 0.29 1.59 L 0.24 0.92 0.84 0.3 1.17 M 0.31 0.71 0.31.23 N 1.1 0.3 1.16 P 0.7 1.01 0.78 0.29 0.99 0.91 0.3 0.24 1.26 Q 0.90.25 1.1 R 1.19 0.31 1.58 S 0.89 0.71 0.51 0.32 T 0.88 0.83 0.29 0.97 V0.73 1.12 0.3 1.08 W 0.26 0.39 1.55 Y 0.87 1.1 0.25 0.77 0.64 1.2 RegionCDR1 FR2 Kabat numbering 28 29 30 31 32 33 34 45 Original amino N R N TY L H Q acid A 0.25 0.63 0.5 0.24 0.85 1.06 D 0.19 0.41 0.34 0.23 0.230.17 0.22 0.77 E 0.4 0.44 0.49 0.72 0.23 0.75 F 0.46 1.1 0.29 0.78 0.27G 0.46 0.67 0.47 1.02 H 0.42 0.98 I 0.44 0.37 0.61 0.97 0.83 0.65 K 0.441.65 1.04 2.17 L 0.39 0.56 0.7 0.59 M 0.39 0.8 0.93 0.35 N 0.32 0.65 P0.36 0.31 0.31 0.31 0.24 0.3 0.34 Q 0.37 0.87 0.25 0.86 R 1.86 0.2 S0.32 0.68 0.29 0.78 T 0.45 0.63 0.29 0.89 V 0.36 0.34 0.61 1.05 0.85 W0.41 0.99 0.24 Y 0.26 0.69 1.04 0.59 Region CDR2 FR3 CDR3 Kabatnumbering 50 51 52 53 54 55 56 74 77 89 Original amino K V S N R F S K RG acid A 0.23 0.93 0.61 0.69 1.13 1.16 1.13 D 0.22 0.33 0.63 0.34 0.360.65 0.77 0.33 E 0.24 0.64 0.54 0.58 0.72 0.71 0.26 F 0.69 1.32 1.09 G0.16 0.84 0.76 0.67 1.31 0.92 H 1.18 0.94 1.05 0.7 I 0.81 0.5 0.82 0.991.07 K 1.08 1.33 1.46 0.4 L 0.24 0.56 0.76 1.02 0.94 M 0.62 0.8 1.050.52 N 0.98 0.92 0.8 P 0.3 0.32 0.33 0.81 0.84 1.16 0.95 0.35 Q 0.181.05 0.77 0.68 0.91 1.04 0.38 R 0.5 1.58 1.31 1.36 0.19 S 0.23 0.69 0.790.69 0.92 T 0.19 0.56 0.65 0.41 0.97 0.84 1.03 V 0.56 0.71 0.95 1.63 W0.81 0.78 0.69 1.38 0.5 Y 0.24 1.12 0.67 0.92 1.46 1.19 Region CDR3 FR4Kabat numbering 90 91 92 93 94 95 96 97 107 Original amino Q G T Q V P YT K acid A 0.5 0.27 0.63 0.85 1.05 0.63 D 0.19 0.16 0.18 0.72 0.89 0.240.17 E 0.86 0.16 0.17 0.75 0.5 0.39 0.17 0.94 F 0.71 1.17 G 0.48 0.37 H0.78 0.23 I 0.34 0.66 K 0.57 L 0.42 0.44 0.24 0.32 M 0.44 N 1.05 P 0.270.27 0.26 0.25 1.26 0.31 Q 0.76 R 1.13 0.66 S 0.73 0.26 0.96 0.96 0.930.43 T 0.26 0.93 V W 0.58 Y 0.17 0.17 0.33 0.87 0.63

TABLE 7 Region CDR1 Kabat numbering 24 25 26 27 27a 27b 27c 27d 27eOriginal amino R S S Q S L V H S acid Affinity up 24 25 26 27 27a 27b27c 27d 27e A 1 0.73 2.57 1.01 D 0.83 8.86 1.06 0.89 0.94 25.07 3.2113640.76 1.23 E 0.89 6.54 0.9 0.99 0.94 26.75 1.1 42.28 1.04 F 2.67 2.051.16 G 0.92 0.8 3.51 1.03 H 1.09 I 0.67 0.87 1.17 1.03 7.77 1.05 K 3.81.32 L 4.93 0.86 0.81 3.37 1.06 M 1.6 1.31 3.43 1.11 N 0.98 3.43 1.01 P0.34 0.79 0.67 2.16 1.01 0.96 3.71 9.21 1.06 Q 0.87 7.48 1.08 R 1.062.35 1.35 S 0.97 0.9 3.04 3.05 T 1.03 0.75 12973.38 0.98 V 0.74 1.11353.86 0.95 W 23.6 1.86 1.32 Y 0.94 0.93 22.2 1.25 1.98 1.1 Domain CDR1FR2 Kabat numbering 28 29 30 31 32 33 34 45 Amino acid N R N T Y L H Qbefore substitution Affinity up 28 29 30 31 32 33 34 45 A 4.18 1.15 1.1666.77 0.82 1.18 D 4455.11 1.58 3.82 30.86 25.92 37.53 2100.2 0.56 E 5.472.83 1.59 0.83 8.03 1.01 F 2.59 f 4.51 0.65 3.5 G 2.41 0.62 2.1 1.08 H 31.08 I 2.81 1.6 1.24 1.1 0.86 0.89 K 2.34 1.35 0.88 4.1 L 3.34 0.9 1.191.03 M 3.29 1.2 0.9 3.16 N 4.46 2.84 P 4.18 14.01 12.14 10.82 61.9832.66 1.22 Q 3.48 1 4.6 0.98 R 1.73 85764 S 4.3 1.05 10.64 1.24 T 2.671.02 12.72 1.1 V 3.73 2.25 2.62 1.26 1.04 W 3.17 0.97 8.45 Y 3.89 1.081.03 2.44 Region CDR2 FR3 CDR3 Kabat numbering 50 51 52 53 54 55 56 7477 89 Original amino K V S N R F S K R G acid Affinity up 50 51 52 53 5455 56 74 77 89 A 59.5 0.9 0.82 0.85 1.16 1.18 1.1 D 114 1.5 0.94 2.8 1.81.02 1.11 1.96 E 57.2 0.88 2.47 0.84 0.92 0.91 34.63 F 0.96 1.12 3.34 G42.4 0.83 1.33 0.88 1.15 0.99 H 1.31 1.02 0.96 1.44 I 0.69 2.69 1.281.01 1.11 K 1.05 1.22 1.21 1.8 L 36.4 1.62 1.43 1.03 2.38 M 1.21 1.290.93 1.96 N 0.91 0.9 1.2 P 27.7 6 7.38 0.98 1.05 1.15 0.98 1.8 Q 8.131.01 1.28 1.04 1.09 0.97 2.11 R 1.83 1.56 1.27 1.15 4127.4 S 45.3 0.880.78 1.15 0.94 T 25.1 2.68 0.89 2.42 1.01 0.85 1.1 V 2.14 1.12 0.94 1.4W 1.01 0.65 1.72 1.12 2.2 Y 195 1.02 0.99 1.13 1.1 1.12 Region CDR3 FR4Kabat numbering 90 91 92 93 94 95 96 97 107 Original amino Q G T Q V P YT K acid Affinity up 90 91 92 93 94 95 96 97 107 A 0.89 28.14 1.35 0.651.05 0.87 D 11.13 44.76 11.19 0.72 1.05 2.37 40.88 E 0.91 48.54 19.561.05 1.18 1.01 46.81 0.95 F 1.75 0.86 G 2.59 1.94 H 1.16 80.34 I 1.911.46 K 0.91 L 1.61 3.06 11.66 1.84 M 2.74 N 0.96 P 15.86 23.05 26.7139.54 1.1 3.35 Q 1.1 R 0.79 1.11 S 0.96 72076 0.81 0.75 0.81 1.19 T39.87 1.06 V W 1.81 Y 36.29 33.84 2.55 0.76 2.45

(6-4) Evaluation of Binding of One-Amino Acid Alteration Antibody to ECM(Extracellular Matrix)

ECM (extracellular matrix) is an extracellular constituent and residesat various sites in vivo. Therefore, an antibody strongly binding to ECMis known to have poorer kinetics in blood (shorter half-life)(WO2012093704 A1). Thus, amino acids that do not enhance ECM binding arepreferably selected as the amino acids that appear in the antibodylibrary.

Each antibody was obtained as an H chain or L chain altered form by themethod described in the paragraph (6-2). Next, its ECM binding wasevaluated according to the method of Reference Example 2. The ECMbinding value (ECL reaction) of each altered form was divided by the ECMbinding value of the antibody MRA (H chain: SEQ ID NO: 57, L chain: SEQID NO: 58) obtained in the same plate or at the same execution date, andthe resulting value is shown in Tables 8 (H chain) and 9 (L chain). Asshown in Tables 8 and 9, some alterations were confirmed to havetendency to enhance ECM binding.

Of the values shown in Tables 8 (H chain) and 9 (L chain), an effectivevalue up to 10 times was adopted to the dual Fab library inconsideration of the effect of enhancing ECM binding by a plurality ofalterations.

TABLE 8 Region FR1 CDR1 FR2 CDR2 Kabat numbering 11 16 19 28 29 30 31 3233 35 43 50 51 52 52a Original amino V R R T F S N A W H K Q I K A acidA D 0.91 1.11 1.1 1.06 4.75 1.07 1.66 E 1.14 1.04 1.8 1.08 4.55 1.181.19 F 2.62 G 3.32 8.82 4.72 H I 2.51 K 41.37 L 3.41 M 4.69 N 3.06 P51.18 Q 1.55 2 R 71.66 11.19 S 2.32 0.95 3.34 T 1.17 3.49 V 17.13 7.32 W8.8 Y Region CDR2 Kabat numbering 52b 52c 53 54 55 56 57 58 59 60 61 6264 65 Original amino K S N N Y A T Y Y A E S K G acid A 2.95 4.5 4.675.82 7.23 2.08 D 2.77 4.02 3.23 4.4 1.23 0.91 E 2.33 4.36 2.75 1.33 2.13F 10.46 15.18 G 5.41 4.43 H I K 58.7 85.86 32.07 16.29 4.07 L 4.07 6.023.56 M N 4.07 4.49 P 9.99 3.83 Q 4.99 3.18 3.23 9.29 1.91 R 7.28 S 3.714.33 6.58 1.89 T V 3.23 W 23.56 Y 19.56 17.47 Region FR3 CDR3 Kabatnumbering 72 73 74 75 76 77 78 82a 95 96 97 Original amino D D S K N S LN V H Y acid A 22.3 D 1.12 0.96 0.65 E 0.76 F G H I K L M N P Q R 2.92 S1.93 T 1.2 2.31 V W Y Region CDR3 FR4 Kabat numbering 98 99 100 100a100b 100c 101 102 105 Original amino G A Y Y G V D A Q acid A 2.7 1.4666.85 D 0.98 1.18 E 1.2 0.3 1.33 F 16.97 2.81 G 1 2.61 56.66 H 2.1216.16 I 63.16 6.63 K 32.29 57.13 8.2 10.3 38.94 L 6.94 M 123.87 N 90.66P 3 Q 2.99 2.12 0.94 130.29 R 48.83 S 2.41 3.34 1 58.7 T 1.6 2.54 V48.47 6.29 W 10.83 Y 27.01 30.37 2.82

TABLE 9 Region CDR1 FR2 Kabat numbering 24 25 26 27 27a 27b 27c 27d 27e28 29 30 31 32 33 34 45 Original amino R S S Q S L V H S N R N T Y L H Qacid A 2.62 2.28 3.25 0.87 2.21 5.92 2.61 D 1.86 1.01 1.31 1.3 1.03 1.160.76 0.64 0.66 0.98 0.6 0.99 E 2.02 1.16 1.22 1.24 1.12 1.04 0.72 1.190.79 1.45 1.16 F 16.43 5.79 1.55 G 1.53 10.04 5.42 3.9 H 13.64 8.6 I11.11 2.68 56.75 4.28 2.87 4.74 K 34.74 31.93 59.62 84.66 L 11.8 3.165.89 M 6.53 3.32 19.75 N 48.45 4.63 P 2.83 2.3 2.7 7.26 Q 1.26 2.58 3.452.31 R 18.19 74.03 69.62 S 2.65 3.3 2.17 T 1.8 2.7 2.32 0.63 4.51 V 2.822.31 2.68 6.43 W 46.73 11.21 Y 1.89 42.7 30.66 3.08 Region CDR2 FR3 CDR3Kabat numbering 50 51 52 53 54 55 56 74 77 89 Original amino K V S N R FS K R G acid A 0.83 3.65 1.78 2.41 16.89 8.43 D 0.64 1.01 1.44 E 0.950.94 F 10.25 31.93 G 0.67 3.5 1.19 6.49 3.26 H 4.88 7.39 6.83 I 2.1123.25 5.1 14.58 K 19.31 5.18 31.33 L 5.65 18.53 M 12.14 5.15 N 7.83 3.964.96 P 4.72 5.49 5.16 6 Q 0.76 2.37 1.33 35.06 2.96 R 34.13 16.5 19.76 S0.84 2.37 T 1.03 1.39 2.48 2.05 6.79 V 4 26.88 W 2.19 26.63 Y 0.88 6.286.18 3.87 28.25 Region CDR3 FR4 Kabat numbering 90 91 92 93 94 95 96 97107 Original amino Q G T Q V P Y T K acid A 3.14 3.11 3.34 3.22 D 1.870.8 0.84 0.88 4.38 0.66 E 2.55 1 0.67 0.85 3.71 0.59 2.96 F 3.44 G H I KL M N 3.01 P 10.7 Q R 44.29 S 4.37 3.12 3.82 3.78 T 2.63 V W Y 3.76 3.262.96 14.49

(6-5) Study on Insertion Site and Length of Peptide for EnhancingDiversity of Library

Example 5 showed that a peptide can be inserted to each site using a GGSsequence without canceling binding to CD3 (CD3ε). If loop extension ispossible for the dual Fab library, the resulting library might includemore types of molecules (or have larger diversity) and permit obtainmentof Fab domains binding to diverse second antigens. Thus, in view ofpresumed reduction in binding activity caused by peptide insertion,V11L/D72A/L78I/D101Q alteration to enhance binding activity against CD3εwas added to the CE115HA000 sequence, which was further linked topE22Hh. A molecule was prepared by the insertion of the GGS linker tothis sequence, as in Example 5, and evaluated for its CD3 binding. TheGGS sequence was inserted between Kabat numbering positions 99 and 100.The antibody molecule was expressed as a one-arm antibody. Specifically,the GGS linker-containing H chain mentioned above and Kn010G3 (SEQ IDNO: 56) were used as H chains, and GLS3000 (SEQ ID NO: 53) linked to thekappa sequence (SEQ ID NO: 55) was adopted as an L chain. Thesesequences were expressed and purified according to Reference Example 1.

(6-6) Confirmation of Binding of GGS Peptide-Inserted CE115 Antibody toCD3

The binding of the GGS peptide-inserted altered antibody to CD3ε wasconfirmed using Biacore by the method described in Example 6. As shownin Table 10, the results demonstrated that the GGS linker can beinserted to loops. Particularly, the GGS linker was able to be insertedto the H chain CDR3 region, which is important for antigen binding, andthe binding to CD3ε was maintained as a result of any of the 3-, 6-, and9-amino acid insertions. Although this study was conducted using the GGSlinker, an antibody library in which various amino acids other than GGSappear may be acceptable.

TABLE 10 Inserted amino  CD3_KD acid sequence [M] GGS  6.31E−08 GGSGGS 3.46E−08 (SEQ ID NO: 90) GGSGGS 3.105E−08 (SEQ ID NO: 90) GGSGGGS4.352E−08 (SEQ ID NO: 92) GGSGGGS 3.429E−08 (SEQ ID NO: 92) GGGSGGGS4.129E−08 (SEQ ID NO: 93) GGGSGGGS 3.753E−08 (SEQ ID NO: 93) GGSGGSGGS 4.39E−08 (SEQ ID NO: 91) GGSGGSGGS 3.537E−08 (SEQ ID NO: 91)No insertion 6.961E−09 CE115HA000 1.097E−07

(6-7) Study on Insertion for Library to H Chain CDR3 Using NNSNucleotide Sequence

The paragraph (6-6) showed that the 3, 6, or 9 amino acids can beinserted using the GGS linker, and inferred that a library having the3-, 6-, or 9-amino acid insertion can be prepared to obtain an antibodybinding to the second antigen by use of a usual antibody obtainmentmethod typified by the phage display method. Thus, a study was conductedon whether the 6-amino acid insertion to CDR3 could maintain binding toCD3 even if various amino acids appeared at the 6-amino acid insertionsite using an NNS nucleotide sequence (which allows every type of aminoacid to appear). In view of presumed reduction in binding activity,primers were designed using the NNS nucleotide sequence such that 6amino acids were inserted between positions 99 and 100 (Kabat numbering)in CDR3 of a CE115HA340 sequence (SEQ ID NO: 59) having higherCD3ε-binding activity than that of CE115HA000. The antibody molecule wasexpressed as a one-arm antibody. Specifically, the altered H chainmentioned above and Kn010G3 (SEQ ID NO: 56) were used as H chains, andGLS3000 (SEQ ID NO: 53) linked to the kappa sequence (SEQ ID NO: 55) wasadopted as an L chain. These sequences were expressed and purifiedaccording to Reference Example 1. The obtained altered antibody wasevaluated for its binding by the method described in the paragraph(6-3). The results are shown in Table 11. The results demonstrated thatthe binding activity against CD3 (CD3ε) is maintained even if variousamino acids appear at the site extended with the amino acids. Table 12shows results of further evaluating the presence or absence ofenhancement in nonspecific binding by the method described in ReferenceExample 2. As a result, the binding to ECM was enhanced if the extendedloop of CDR3 was rich in amino acids having a positively charged sidechain. Therefore, it was desired that three or more amino acids having apositively charged side chain should not appear in the loop.

TABLE 11 CDR3 VH CD3_KD[M] 9 1 0 CE115HA340 2.0E−08 5 6 7 8 9 0 a b c de f g h i k 1 1 2 CE115HA340 2.7E−08 V H Y A A X X X X X X Y Y G V — — DA NNS6f17 7.4E−08 . . . . . W G E G V V . . . . . . . . NNS6f27 3.8E−08. . . . . V W G S V W . . . . . . . . NNS6f29 9.0E−08 . . . . . I Y Y PT N . . . . . . . . NNS6f47 3.1E−08 . . . . . H F M W W G . . . . . . .. NNS6f50 7.1E−08 . . . . . L T G G L G . . . . . . . . NNS6f51 3.1E−08. . . . . G F L V L W . . . . . . . . NNS6f52 5.2E−08 . . . . . Y M L GL G . . . . . . . . NNS6f54 2.9E−08 . . . . . F E W V G W . . . . . . .. NNS6f55 3.1E−08 . . . . . A G R W L A . . . . . . . . NNS6f56 2.1E−08. . . . . R E A T R W . . . . . . . . NNS6f58 4.4E−08 . . . . . S W Q VS R . . . . . . . . NNS6f59 2.0E−07 . . . . . L L V Q E G . . . . . . .. NNS6f62 6.1E−08 . . . . . N G G T R H . . . . . . . . NNS6f63 6.9E−08. . . . . G G G G W V . . . . . . . . NNS6f64 7.8E−08 . . . . . L V S LT V . . . . . . . . NNS6f67 3.6E−08 . . . . . G L L R A A . . . . . . .. NNS6f68 4.5E−08 . . . . . V E W G R W . . . . . . . . NNS6f71 5.1E−08. . . . . G W V L G S . . . . . . . . NNS6f72 1.5E−07 . . . . . E G I WW G . . . . . . . . NNS6f73 2.6E−08 . . . . . W V V G V R . . . . . . ..

TABLE 12 CDR3 ECL reaction Ratio 9 1 0 H chain ECM 3 μg/ml MRA ECM vsMRA 5 6 7 8 9 0 a b c d e f g h i k 1 1 2 CE115HA340 394 448 03 V H Y AA X X X X X X Y Y G V — — D A NNS6f17 409 448 0.9 . . . . . W G E G V V. . . . . . . . NNS6f27 3444 448 7.7 . . . . . V W G S V W . . . . . . .. NNS6f29 481 448 1.1 . . . . . I Y Y P T N . . . . . . . . NNS6f4794137 448 210.3 . . . . . H F M W W G . . . . . . . . NNS6f50 385 5640.7 . . . . . L T G G L G . . . . . . . . NNS6f51 20148 564 35.7 . . . .. G F L V L W . . . . . . . . NNS6f52 790 564 1.4 . . . . . Y M L G L G. . . . . . . . NNS6f54 1824 564 3.2 . . . . . F E W V G W . . . . . . .. NNS6f55 14183 564 25.1 . . . . . A G R W L A . . . . . . . . NNS6f566534 564 11.6 . . . . . R E A T R W . . . . . . . . NNS6f58 2700 564 4.8. . . . . S W Q V S R . . . . . . . . NNS6f59 388 564 0.7 . . . . . L LV Q E G . . . . . . . . NNS6f62 554 564 1.0 . . . . . N G G T R H . . .. . . . . NNS6f63 624 564 1.1 . . . . . G G G G W V . . . . . . . .NNS6f64 603 564 1.1 . . . . . L V S L T V . . . . . . . . NNS6f67 1292564 2.3 . . . . . G L L R A A . . . . . . . . NNS6f68 2789 564 4.9 . . .. . V E W G R W . . . . . . . . NNS6f71 618 564 11 . . . . . G W V L G S. . . . . . . . NNS6f72 536 564 0.9 . . . . . E G I W W G . . . . . . .. NNS6f73 2193 564 3.9 . . . . . W V V G V R . . . . . . . .

(6-7) Design and Construction of Dual Fab Library

On the basis of the study described in Example 6, an antibody library(dual Fab library) for obtaining an antibody binding to CD3 and thesecond antigen was designed as follows:

step 1: selecting amino acids that maintain the ability to bind to CD3(CD3ε) (to secure 80% or more of the amount of CE115HA000 bound to CD3);step 2: selecting amino acids that keep ECM binding within 10 times thatof MRA compared with before alteration; andstep 3: inserting 6 amino acids to between positions 99 and 100 (Kabatnumbering) in H chain CDR3.

The antigen-binding site of Fab can be diversified by merely performingthe step 1. The resulting library can therefore be used for identifyingan antigen-binding molecule binding to the second antigen. Theantigen-binding site of Fab can be diversified by merely performing thesteps 1 and 3. The resulting library can therefore be used foridentifying an antigen-binding molecule binding to the second antigen.Even library design without the step 2 allows an obtained molecule to beassayed and evaluated for ECM binding.

Thus, for the dual Fab library, sequences derived from CE115HA000 byadding the V11L/L78I mutation to FR (framework) and further diversifyingCDRs as shown in Table 13 were used as H chains, and sequences derivedfrom GLS3000 by diversifying CDRs as shown in Table 14 were used as Lchains. These antibody library fragments can be synthesized by a DNAsynthesis method generally known to those skilled in the art. The dualFab library may be prepared as (1) a library in which H chains arediversified as shown in Table 13 while L chains are fixed to theoriginal sequence GLS3000 or the L chain having enhanced CD3ε bindingdescribed in Example 6, (2) a library in which H chains are fixed to theoriginal sequence (CE115HA000) or the H chain having enhanced CD3εbinding described in Example 6 while L chains are diversified as shownin Table 14, and (3) a library in which H chains are diversified asshown in Table 13 while L chains are diversified as shown in Table 14.The H chain library sequences derived from CE115HA000 by adding theV11L/L78I mutation to FR (framework) and further diversifying CDRs asshown in Table 13 were entrusted to the DNA synthesizing company DNA2.0,Inc. to obtain antibody library fragments (DNA fragments). The obtainedantibody library fragments were inserted to phagemids for phage displayamplified by PCR. GLS3000 was selected as L chains. The constructedphagemids for phage display were transferred to E. coli byelectroporation to prepare E. coli harboring the antibody libraryfragments.

TABLE 13 CDR3 CDR2 Kabat numbering 3 5 5 6 1 2 3 4 5 0 1 2 a b c 3 4 5 67 8 9 0 1 2 3 4 5 Pre- N A W M H Q I K A K S N N Y A T Y Y A E S V K Gsubstitution Library I A W M H Q I K D R A Q A Y L A Y Y A P S V K G N KG S G N N E S L N L A T Q Q V S S N CDR3 Kabat numbering 9 1 0 5 6 7 8 90 a b c d e f g h i 1 2 Pre- V H Y G A x x x x x x Y Y G M D Isubstitution Library V H Y A A A A G A L P A Y G V D I G L V V S V G G SF P S G G T L S S Q G S Q S S Y G Y Y K T T T T F S F F Y Q Y Y D Y G HF F F D

TABLE 14 Domain CDR1 FR2 CDR2 Kabat numbering 2 3 4 5 4 5 6 7 a b c d e8 9 0 1 2 3 4 5 0 Before R S S Q S L V H S N R N T Y L H Q Ksubstitution Library R S S O S L V H S N R N T Y L H Q K A A D D E I L AF I A A E P E P P G H I G G T V I M V T Q L Q V S M Y T N Y P Q T VDomain CDR2 FR3 CDR3 FR4 Kabat numbering 7 9 10 1 2 3 4 5 6 4 7 9 0 1 23 4 5 6 7 7 Before V S N R F S K R G Q G T Q V P Y T K substitutionLibrary V S N R F S K R G Q G T Q V P Y T K G A P P A G T S A E S A A FE I Q W G H N S D Y H I T N K L S N M T P N Y P Q T V Y

[Example 7] Obtainment of Fab Domain Binding to CD3 and Second Antigen(IL6R) from Dual Fab Library

(7-1) Obtainment of Fab Domain Binding to Human IL6R

Fab domains (antibody fragments) binding to human IL6R were identifiedfrom the dual Fab library designed and constructed in Example 6.Biotin-labeled human IL6R was used as an antigen, and antibody fragmentshaving the ability to bind to human IL6R were enriched.

Phages were produced from the E. coli harboring the constructedphagemids for phage display. 2.5 M NaCl/10% PEG was added to the culturesolution of the E. coli that had produced phages, and a pool of thephages thus precipitated was diluted with TBS to obtain a phage librarysolution. Next, BSA (final concentration: 4%) was added to the phagelibrary solution. The panning method was performed with reference to ageneral panning method using antigens immobilized on magnetic beads (J.Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247(1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; and Mol. CellProteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution and thereby contacted with the phagelibrary solution at room temperature for 60 minutes. After addition ofBSA-blocked magnetic beads, the antigen-phage complexes were attached tothe magnetic beads at room temperature for 15 minutes. The beads werewashed three times with TBST (TBS containing 0.1% Tween 20; TBS wasavailable from Takara Bio Inc.) and then further washed twice with 1 mLof TBS. After addition of 0.5 mL of 1 mg/mL trypsin, the beads weresuspended at room temperature for 15 minutes, immediately after whichthe beads were separated using a magnetic stand to recover a phagesolution. The recovered phage solution was added to 10 mL of an E. colistrain ER2738 in a logarithmic growth phase (OD600: 0.4-0.5). The E.coli strain was infected by the phages through the gentle spinnerculture of the strain at 37° C. for 1 hour. The infected E. coli wasinoculated to a plate of 225 mm×225 mm. Next, phages were recovered fromthe culture solution of the inoculated E. coli to prepare a phagelibrary solution. This cycle, called panning, was repeated severaltimes. In the second and subsequent rounds of panning, 40 pmol of thebiotin-labeled antigen was used. In the fourth round of panning, thephages were enriched with binding ability against CD3 as an index.Specifically, 250 pmol of biotin-labeled CD3ε peptide antigen (aminoacid sequence: SEQ ID NO: 60) was added to the prepared phage librarysolution and thereby contacted with the phage library solution at roomtemperature for 60 minutes. After addition of BSA-blocked magneticbeads, the antigen-phage complexes were attached to the magnetic beadsat room temperature for 15 minutes. The beads were washed with 1 mL ofTBS containing 0.1% Tween 20 and TBS. The beads supplemented with 0.5 mLof 1 mg/mL trypsin were suspended at room temperature for 15 minutes,immediately after which the beads were separated using a magnetic standto recover a phage solution. The phages recovered from thetrypsin-treated phage solution were added to 10 mL of an E. coli strainER2738 in a logarithmic growth phase (OD600: 0.4-0.7). The E. colistrain was infected by the phages through the gentle spinner culture ofthe strain at 37° C. for 1 hour. The infected E. coli was inoculated toa plate of 225 mm×225 mm. Next, phages were recovered from the culturesolution of the inoculated E. coli to recover a phage library solution.

In order to prevent a plurality of phages from infecting one E. coli, aphage library solution prepared from E. coli infected by phagesrecovered by the fifth round of panning was diluted again 100,000-fold,and E. coli was infected by the resulting phage solution to obtainsingle colonies.

(7-2) Binding of Fab Domain Displayed by Phage to CD3 or IL6R (PhageELISA)

A phage-containing culture supernatant was recovered according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from each singlecolony of the E. coli obtained by the method described above. Afteraddition of BSA (final concentration: 4%), the phage-containing culturesupernatant was subjected to ELISA by the following procedures:StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd.) was coatedovernight at 4° C. or at room temperature for 1 hour with 100 μL of PBScontaining the biotin-labeled antigen (biotinylated CD3ε peptide orbiotinylated human IL6R). Each well of the plate was washed with PBST toremove unbound antigens. Then, the well was blocked with 250 μL of 4%BSA-TBS for 1 hour or longer. After removal of 4% BSA-TBS, the preparedculture supernatant was added to each well, and the plate was leftstanding at room temperature for 1 hour so that the phage-displayedantibody bound to the antigen contained in each well. Each well waswashed with TBST, and HRP-conjugated anti-M13 antibodies (AmershamPharmacia Biotech Inc.) diluted with TBS containing BSA (finalconcentration: 4%) were then added to each well. The plate was incubatedfor 1 hour. After washing with TBST, TMB single solution (ZYMEDLaboratories, Inc.) was added to the well. The chromogenic reaction ofthe solution in each well was terminated by the addition of sulfuricacid. Then, the developed color was assayed on the basis of absorbanceat 450 nm. The results are shown in FIG. 19. Clones #50 and #62 wereshown to have binding activity against CD3ε and human IL6R. In otherwords, clones exhibiting binding activity against the second antigen (inExample 7, human IL6R) were successfully selected by use of the dual Fablibrary. Clones exhibiting binding activity can be selected from afurther increased number of library members to be evaluated, thenconverted to IgG (the VH and VL sequences of each clone are linked tohuman H chain and L chain constant domains, respectively), and evaluatedfor their binding activity against CD3ε and the second antigen (humanIL6R). Furthermore, whether or not to bind to CD3ε and the secondantigen (human IL6R) at the same time can be examined by the methoddescribed in Example 3 or 4 or the competition method. The competitionmethod shows that the antibody does not bind to CD3ε and the secondantigen at the same time, for example, when the level of its binding toCD3ε is reduced in the presence of the second antigen as compared withthe antibody alone.

[Example 8] Obtainment of Fab Domain Binding to CD3 and Second Antigen(Human IgA) from Dual Fab Library

(8-1) Obtainment of Fab Domain Binding to Human IgA

IgA, which is an in vivo abundant antibody isotype, is known as amolecule involved in intestinal or mucosal biophylaxis and also known tobind to FcaR (Fc alpha receptor) (J. Pathol. 208: 270-282, 2006).

Fab domains (antibody fragments) binding to human IgA were identifiedfrom the dual Fab library designed and constructed in Example 6.Biotin-labeled human IgA (described in Reference Example 3) was used asan antigen, and antibody fragments having the ability to bind to humanIgA were enriched.

Phages were produced from the E. coli harboring the constructedphagemids for phage display. 2.5 M NaCl/10% PEG was added to the culturesolution of the E. coli that had produced phages, and a pool of thephages thus precipitated was diluted with TBS to obtain a phage librarysolution. Next, BSA (final concentration: 4%) was added to the phagelibrary solution. The panning method was performed with reference to ageneral panning method using antigens immobilized on magnetic beads (J.Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247(1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; and Mol. CellProteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution and thereby contacted with the phagelibrary solution at room temperature for 60 minutes. After addition ofBSA-blocked magnetic beads, the antigen-phage complexes were attached tothe magnetic beads at room temperature for 15 minutes. The beads werewashed three times with TBST (TBS containing 0.1% Tween 20; TBS wasavailable from Takara Bio Inc.) and then further washed twice with 1 mLof TBS. After addition of 0.5 mL of 1 mg/mL trypsin, the beads weresuspended at room temperature for 15 minutes, immediately after whichthe beads were separated using a magnetic stand to recover a phagesolution. The recovered phage solution was added to 10 mL of an E. colistrain ER2738 in a logarithmic growth phase (0D600: 0.4-0.5). The E.coli strain was infected by the phages through the gentle spinnerculture of the strain at 37° C. for 1 hour. The infected E. coli wasinoculated to a plate of 225 mm×225 mm. Next, phages were recovered fromthe culture solution of the inoculated E. coli to prepare a phagelibrary solution. This cycle, called panning, was repeated 4 times. Inthe second and subsequent rounds of panning, 40 pmol of human IgA wasused.

(8-2) Binding of Fab Domain Displayed by Phage to CD3 or Human IgA

A phage-containing culture supernatant was recovered according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from each singlecolony of the E. coli obtained by the method described above. Afteraddition of BSA (final concentration: 4%), the phage-containing culturesupernatant was subjected to ELISA by the following procedures:StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd.) was coatedovernight at 4° C. or at room temperature for 1 hour with 100 μL of PBScontaining the biotin-labeled antigen (biotin-labeled CD3ε peptide orbiotin-labeled human IgA; Reference Example 3). Each well of the platewas washed with PBST to remove unbound antigens. Then, the well wasblocked with 250 μL of 0.1×TBS/150 mM NaCl/0.02% skim milk for 1 hour orlonger. After removal of 0.1×TBS/150 mM NaCl/0.02% skim milk, theprepared culture supernatant was added to each well, and the plate wasleft standing at room temperature for 1 hour so that the phage-displayedantibody bound to the antigen contained in each well. Each well waswashed with 0.1×TBS/150 mM NaCl/0.01% Tween 20, and HRP-conjugatedanti-M13 antibodies (Amersham Pharmacia Biotech Inc.) diluted with0.1×TBS/150 mM NaCl/0.01% Tween 20 were then added to each well. Theplate was incubated for 1 hour. After washing with TBST, TMB singlesolution (ZYMED Laboratories, Inc.) was added to the well. Thechromogenic reaction of the solution in each well was terminated by theaddition of sulfuric acid. Then, the developed color was assayed on thebasis of absorbance at 450 nm. The results are shown in FIG. 20. Asshown in FIG. 20, some clones were shown to bind to CD3 and human IgA.Thus, clones exhibiting binding activity against the second antigen (inExample 8, human IgA) were successfully selected by use of the dual Fablibrary.

(8-3) Binding of IgG Having Obtained Fab Domain to CD3 or Human IgA

The VH fragment of each clone shown to bind to CD3 and human IgA in theparagraph (8-2) was amplified from E. coli having the sequence by PCRusing primers specifically binding to the H chain in the dual Fablibrary. The amplified VH fragment was integrated to pE22Hh-containingplasmids for expression in animal cells by the method of ReferenceExample 1, and expressed and purified as a one-arm antibody, as inExample 6(6-2). The name of each clone and SEQ ID NO of its H chainsequence are shown in Table 15. Specifically, each H chain shown inTable 15 and Kn010G3 (SEQ ID NO: 56) were used as H chains, and GLS3000(SEQ ID NO: 53) linked to the kappa sequence (SEQ ID NO: 55) was adoptedas an L chain. These sequences were expressed and purified according toReference Example 1.

TABLE 15 Sample name SEQ ID NO IGAR4C09#1 61 IGAR4C12#4 62 IGAR6(2)B02#163

The antibody molecule having the obtained Fab region was evaluated forits binding to CD3ε and human IgA by the electrochemiluminescence method(ECL method). Specifically, biotin-labeled CD3ε peptide (described inExample 7) or biotin-labeled human IgA (Reference Example 3) dilutedwith a TBST solution (TBS (manufactured by Takara Bio Inc.) supplementedwith 0.1% Tween 20), each antibody solution adjusted to 2 μg/mL, andanti-human IgG antibody (Invitrogen Corp., #628400) tagged withsulfo-tag were each added at 25 μL/well to Nunc-Immuno™ MicroWell™ 96well round plates (Nunc), and mixed, and the plate was then incubated atroom temperature for 1 hour or longer while shielded from light to forman antibody-antigen complex. A TBST solution containing 0.5% BSA(referred to as a blocking solution) was added at 150 μL/well tostreptavidin plate (MSD K.K.), and the plate was incubated at roomtemperature for 1 hour or longer. After removal of the blockingsolution, each well was washed three times with 250 μL of a TBS(−)solution. The antibody-antigen complex solution was added thereto at 50μL/well, and the plate was incubated at room temperature for 1 hour sothat the biotinylated antigen-antibody-detection sulfo-tag antibodycomplex solution bound via the biotinylated antigen to the streptavidinplate. After removal of the antibody-antigen complex solution, each wellwas washed three times with a TBST solution, and a solution of 4×READbuffer (MSD K.K.) diluted 2-fold with water was added thereto at 150μL/well, followed by the detection of the luminescence signal of thesulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 21. The clones confirmed to bind by phageELISA were each converted to IgG, also including those containing someamino acid mutations. Consequently, these sequences confirmed to bind byphage ELISA were shown to bind to CD3ε and human IgA, even in the formof IgG.

These results demonstrated that: an antibody binding to the secondantigen can be obtained from the dual Fab library; and clones confirmedto bind can be enriched even in the form of Fc region-containing IgG,though only Fab domains are enriched in usual panning using a phagelibrary. Thus, it was concluded that the dual Fab library serves as alibrary that permits obtainment of a Fab domain having the ability tobind to the second antigen while maintaining the ability to bind to CD3.

(8-4) Evaluation of Binding of IgG Having Obtained Fab Domain to CD3(CD3ε) and Human IgA at Same Time

In the paragraph (8-3), the clones obtained from the dual Fab librarywere shown to have binding activity even in the form of IgG. Next, eachobtained IgG was evaluated for its binding to CD3 (CD3ε) and human IgAat the same time by the competition method (electrochemiluminescencemethod (ECL method)). It can be expected that: when the IgG binds to CD3(CD3ε) and human IgA at the same time, the addition of CD3 (CD3ε) toIgA-bound antibodies causes no change in ECL signal; and when the IgGcannot bind to these antigens at the same time, the addition of CD3(CD3ε) decreases the ECL signal due to the binding of some antibodies toCD3 (CD3ε).

Specifically, 25 μL of biotinylated human IgA diluted with a TBSTsolution, 12.5 μL of each antibody solution adjusted to 1 μg/mL, 12.5 μLof TBST or CD3ε homodimer protein (9.4 pmol/μL) for competition, and 25μL of anti-human IgG antibody (Invitrogen Corp., #628400) tagged withsulfo-tag were added to each well of Nunc-Immuno™ MicroWell™ 96 wellround plates (Nunc), and mixed, and the plate was then incubated at roomtemperature for 1 hour or longer while shielded from light to form anantibody-antigen complex. A TBST solution containing 0.5% BSA (referredto as a blocking solution; the TBST solution was TBS (manufactured byTakara Bio Inc.) supplemented with 0.1% Tween 20) was added at 150μL/well to streptavidin plate (MSD K.K.), and the plate was incubated atroom temperature for 1 hour or longer. After removal of the blockingsolution, each well was washed three times with 250 μL of a TBSTsolution. The antibody-antigen complex solution was added thereto at 50μL/well, and the plate was incubated at room temperature for 1 hour sothat the biotinylated antigen-antibody-detection sulfo-tag antibodycomplex solution bound via the biotinylated antigen to the streptavidinplate. After removal of the antibody-antigen complex solution, each wellwas washed three times with a TBST solution, and a solution of 4×READbuffer (MSD K.K.) diluted 2-fold with water was added thereto at 150μL/well, followed by the detection of the luminescence signal of thesulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 22. The addition of the CD3ε homodimerprotein for competition was confirmed to decrease the ECL signal ascompared with the addition of TBST. These results indicated that themolecule binding to CD3 and human IgA, found in this study, is a dualFab molecule that cannot bind to human IgA in a state bound with CD3.There results demonstrated that an antibody having the ability to bindto the second antigen can be obtained from the dual Fab library and,among others, can be obtained as a dual Fab molecule that cannot bind tothe second antigen in a state bound with CD3 (or cannot bind to CD3 in astate bound with the second antigen), i.e., cannot bind to plural typesof antigens at the same time.

It is obvious to those skilled in the art that, provided that a bindingmolecule is found in the binding activity evaluation using phages in theparagraph (8-2), the sequence variety of the binding molecule can beincreased by increasing the number of library members to be evaluated.Thus, it was concluded that the dual Fab library serves as a librarythat permits obtainment of a Fab domain having the ability to bind tothe second antigen while maintaining the ability to bind to CD3. In thisExample, the dual Fab library diversified by only H chains was used. Alarger library size (also called diversity; which means that the libraryincludes diverse sequences) usually allows more antigen-bindingmolecules to be obtained. Therefore, a dual Fab library diversified byboth H and L chains can also be used for obtaining dual Fab molecules,as shown in this Example.

Provided that dual Fab molecules can be prepared as shown in Example 8,Fab or an antigen-binding domain binding to the third antigen can beidentified by a method generally known to those skilled in the art, forexample, a hybridoma method or a method for selecting binding antibodies(or binding domains) from antibody libraries. An antibody having theidentified antigen-binding domain (e.g., Fab) binding to the thirdantigen and the Fab domain of the dual Fab molecule can be obtained as amultispecific antibody by a multispecific antibody preparation methodgenerally known to those skilled in the art, for example, a method forpreparing an antibody having common L chains and two different H chains(technique controlling the interface of each domain in the Fc region),CrossMab, or Fab arm exchange. In other words, provided that a dual Fabmolecule can be identified, the desired multispecific antibody can beobtained according to a method generally known to those skilled in theart by combining Fab binding to the third antigen with the dual Fabbinding to the first and second antigens as shown in Example 8.

(8-5) CD3/Human IgA Dual Fab Molecule

In Example 8, the obtained dual Fab molecule was shown to bind to CD3εand human IgA, but not bind to CD3ε and human IgA at the same time. Anantigen-binding domain binding to the third antigen can be further addedthereto by a method generally known to those skilled in the art.

In recent years, an IgA molecule altered to bind to EGFR, a cancerantigen, has been found to induce the cell death of cancer cellsexpressing EGFR (J Immunol 2007; 179: 2936-2943). As this mechanism, theIgA receptor FcαR is expressed in polymorphonuclear cells and reportedlyinduces the autophagy of cancer cells (J Immunol 2011; 187: 726-732).This Example revealed that the dual Fab molecule binding to CD3 and IgAcan be constructed. Anti-tumor effects mediated by FcαR can be expectedby searching for a molecule binding to FcαR via IgA by a methodgenerally known to those skilled in the art (e.g., ELISA or the ECLmethod). In other words, this dual Fab can induce both of Tcell-mediated cytotoxic activity by binding to CD3ε and cytotoxicactivity mediated by FcαR-expressing cells via binding to IgA againstcells expressing an arbitrary third antigen and can be expected toproduce strong cytotoxic activity.

[Example 9] Obtainment of Fab Domain Binding to CD3 and Second Antigen(Human CD154) from Dual Fab Library

(9-1) Obtainment of Fab Domain Binding to Human CD154

Fab domains (antibody fragments) binding to human CD154 were identifiedfrom the dual Fab library designed and constructed in Example 6.Antibody fragments having the ability to bind to human CD154 wereenriched using biotin-labeled human CD154 as an antigen.

Phages were produced from the E. coli harboring the constructedphagemids for phage display. 2.5 M NaCl/10% PEG was added to the culturesolution of the E. coli that had produced phages, and a pool of thephages thus precipitated was diluted with TBS to obtain a phage librarysolution. Next, BSA (final concentration: 4%) was added to the phagelibrary solution. The panning method was performed with reference to ageneral panning method using antigens immobilized on magnetic beads (J.Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247(1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; and Mol. CellProteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution and thereby contacted with the phagelibrary solution at room temperature for 60 minutes. After addition ofBSA-blocked magnetic beads, the antigen-phage complexes were attached tothe magnetic beads at room temperature for 15 minutes. The beads werewashed three times with TBST (TBS containing 0.1% Tween 20; TBS wasavailable from Takara Bio Inc.) and then further washed twice with 1 mLof TBS. After addition of 0.5 mL of 1 mg/mL trypsin, the beads weresuspended at room temperature for 15 minutes, immediately after whichthe beads were separated using a magnetic stand to recover a phagesolution. The recovered phage solution was added to 10 mL of an E. colistrain ER2738 in a logarithmic growth phase (0D600: 0.4-0.5). The E.coli strain was infected by the phages through the gentle spinnerculture of the strain at 37° C. for 1 hour. The infected E. coli wasinoculated to a plate of 225 mm×225 mm. Next, phages were recovered fromthe culture solution of the inoculated E. coli to prepare a phagelibrary solution. This cycle, called panning, was repeated 5 times. Inthe second and subsequent rounds of panning, 40 pmol of human CD154 wasused.

(9-2) Binding of Fab Domain Displayed by Phage to CD3 or Human CD154

A phage-containing culture supernatant was recovered according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from each singlecolony of the E. coli obtained by the method described above. Afteraddition of BSA (final concentration: 4%), the phage-containing culturesupernatant was subjected to ELISA by the following procedures:StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd.) was coatedovernight at 4° C. or at room temperature for 1 hour with 100 μL of PBScontaining the biotin-labeled antigen (biotin-labeled CD3ε peptide orbiotin-labeled CD154). Each well of the plate was washed with PBST toremove unbound antigens. Then, the well was blocked with 250 μL of0.1×TBS/150 mM NaCl/0.02% skim milk for 1 hour or longer. After removalof 0.1×TBS/150 mM NaCl/0.02% skim milk, the prepared culture supernatantwas added to each well, and the plate was left standing at roomtemperature for 1 hour so that the phage-displayed antibody bound to theantigen contained in each well. Each well was washed with 0.1×TBS/150 mMNaCl/0.01% Tween 20, and HRP-conjugated anti-M13 antibodies (AmershamPharmacia Biotech Inc.) diluted with 0.1×TBS/150 mM NaCl/0.01% Tween 20were then added to each well. The plate was incubated for 1 hour. Afterwashing with TBST, TMB single solution (ZYMED Laboratories, Inc.) wasadded to the well. The chromogenic reaction of the solution in each wellwas terminated by the addition of sulfuric acid. Then, the developedcolor was assayed on the basis of absorbance at 450 nm. The results areshown in FIG. 23. As shown in FIG. 23, some clones were shown to bind toCD3 and CD154. Thus, clones exhibiting binding activity against thesecond antigen (in Example 9, human CD154) were successfully selected byuse of the dual Fab library. As shown in Examples 7, 8, and 9, bindingFab domains can be obtained for 3 different antigens, indicating thatthe dual Fab library functions as a library for obtaining a moleculebinding to the second antigen.

(9-3) Binding of IgG Having Obtained Fab Domain to CD3 or Human CD154

The VH fragment of each clone shown to bind to CD3 and human CD154 inthe paragraph (9-2) was amplified from E. coli having the sequence byPCR using primers specifically binding to the H chain in the dual Fablibrary. The amplified VH fragment was integrated to pE22Hh-containingplasmids for expression in animal cells by the method of ReferenceExample 1, and expressed and purified as a one-arm antibody, as inExample 6(6-2). The name of each obtained sequence and SEQ ID NO of itsH chain sequence are shown in Table 16. Specifically, each H chain shownin Table 16 and Kn010G3 (SEQ ID NO: 56) were used as H chains, andGLS3000 (SEQ ID NO: 53) linked to the kappa sequence (SEQ ID NO: 55) wasadopted as an L chain. These sequences were expressed and purifiedaccording to Reference Example 1.

TABLE 16 Sample name SEQ ID NO 154R3A01#1 64 154R3A01#2 65 154R3A01#3 66154R3A01#4 67 154R3B01#1 68 154R3F02#2 69 154R4B03#1 70 154R4B03#4 71154R4B07#1 72 154R4F08#1 73 154R5A02#1 74 154R5A02#2 75 154R5F08#1 76154R5F08#3 77 154R5E11#3 78

The antibody molecule having the Fab region shown to bind to CD3 andhuman CD154 by phage ELISA in the paragraph (9-2) was evaluated for itsbinding to CD3 and human CD154 by the electrochemiluminescence method(ECL method). Specifically, 25 μL of biotinylated CD3 or biotinylatedhuman CD154 diluted with a TBST solution, 25 μL of each antibodysolution adjusted to 2 μg/mL, and 25 μL of anti-human IgG antibody(Invitrogen Corp., #628400) tagged with sulfo-tag were added to eachwell of Nunc-Immuno™ MicroWell™ 96 well round plates (Nunc), and mixed,and the plate was then incubated at room temperature for 1 hour orlonger while shielded from light to form an antibody-antigen complex. ATBST solution containing 0.5% BSA (referred to as a blocking solution;the TBST solution was TBS (manufactured by Takara Bio Inc.) supplementedwith 0.1% Tween 20) was added at 150 μL/well to streptavidin plate (MSDK.K.), and the plate was incubated at room temperature for 1 hour orlonger. After removal of the blocking solution, each well was washedthree times with 250 μL of a TBST solution. The antibody-antigen complexsolution was added thereto at 50 μL/well, and the plate was incubated atroom temperature for 1 hour so that the biotinylatedantigen-antibody-detection sulfo-tag antibody complex solution bound viathe biotinylated antigen to the streptavidin plate. After removal of theantibody-antigen complex solution, each well was washed three times witha TBST solution, and a solution of 4×READ buffer (MSD K.K.) diluted2-fold with water was added thereto at 150 μL/well, followed by thedetection of the luminescence signal of the sulfo-tag using SectorImager 2400 (MSD K.K.).

The results are shown in FIG. 24. The clones confirmed to bind by phageELISA were each converted to IgG, also including those containing someamino acid mutations. Consequently, these sequences confirmed to bind byphage ELISA were shown to bind to CD3ε and human CD154, even in the formof IgG.

These results demonstrated that: an antibody binding to the secondantigen can be obtained from the dual Fab library; and clones confirmedto bind can be enriched not only for human IgA but for human CD154 evenin the form of Fc region-containing IgG, though only Fab domains areenriched in usual panning using a phage library. Thus, it was concludedthat the dual Fab library serves as a library that permits obtainment ofa Fab domain having the ability to bind to the second antigen whilemaintaining the ability to bind to CD3.

(9-4) Evaluation of Binding of IgG Having Obtained Fab Domain to CD3εand Human CD154 at Same Time

In the paragraph (9-3), the clones obtained from the dual Fab librarywere shown to have binding activity even in the form of IgG. Next, eachobtained IgG was evaluated for its binding to CD3 and human CD154 at thesame time by the competition method (electrochemiluminescence method(ECL method)). It can be expected that: when the IgG binds to CD3 andhuman CD154 at the same time, the addition of CD3 to CD154-boundantibodies causes no change in ECL signal; and when the IgG cannot bindto these antigens at the same time, the addition of CD3 decreases theECL signal due to the binding of some antibodies to CD3.

Specifically, 25 μL of biotinylated human CD154 diluted with a TBSTsolution, 12.5 μL of each antibody solution adjusted to 1 μg/mL, 12.5 μLof TBST or CD3ε homodimer protein (9.4 pmol/μL) for competition, and 25μL of anti-human IgG antibody (Invitrogen Corp., #628400) tagged withsulfo-tag were added to each well of Nunc-Immuno™ MicroWell™ 96 wellround plates (Nunc), and mixed, and the plate was then incubated at roomtemperature for 1 hour or longer while shielded from light to form anantibody-antigen complex. A TBST solution containing 0.5% BSA (referredto as a blocking solution; the TBST solution was TBS (manufactured byTakara Bio Inc.) supplemented with 0.1% Tween 20) was added at 150μL/well to streptavidin plate (MSD K.K.), and the plate was incubated atroom temperature for 1 hour or longer. After removal of the blockingsolution, each well was washed three times with 250 μL of a TBSTsolution. The antibody-antigen complex solution was added thereto at 50μL/well, and the plate was incubated at room temperature for 1 hour sothat the biotinylated antigen-antibody-detection sulfo-tag antibodycomplex solution bound via the biotinylated antigen to the streptavidinplate. After removal of the antibody-antigen complex solution, each wellwas washed three times with a TBST solution, and a solution of 4×READbuffer (MSD K.K.) diluted 2-fold with water was added thereto at 150μL/well, followed by the detection of the luminescence signal of thesulfo-tag using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 25. The addition of the CD3ε homodimerprotein for competition was confirmed to decrease the ECL signal ascompared with the addition of TBST. These results indicated that themolecule binding to CD3ε and human CD154, found in this study, is a dualFab molecule that cannot bind to human CD154 in a state bound with CD3.There results demonstrated that an antibody binding to the secondantigen can be obtained from the dual Fab library and, among others, canbe obtained as a dual Fab molecule that cannot bind to the secondantigen in a state bound with CD3 (or cannot bind to CD3 in a statebound with the second antigen), i.e., cannot bind to plural types ofantigens at the same time.

It is obvious to those skilled in the art that, provided that a bindingmolecule is found in the binding activity evaluation using phages in theparagraph (9-2), the sequence variety of the binding molecule can beincreased by increasing the number of library members to be evaluated.Thus, it was concluded that the dual Fab library serves as a librarythat permits obtainment of a Fab domain having the ability to bind tothe second antigen while maintaining the ability to bind to CD3. In thisExample, the dual Fab library diversified by only H chains was used. Alarger library size (also called diversity; which means that the libraryincludes diverse sequences) usually allows more antigen-bindingmolecules to be obtained. Therefore, a dual Fab library diversified byboth H and L chains can also be used for obtaining dual Fab molecules,as shown in this Example.

Provided that dual Fab molecules can be prepared as shown in Example 9,Fab or an antigen-binding domain binding to the third antigen can beidentified by a method generally known to those skilled in the art, forexample, a hybridoma method or a method for selecting binding antibodiesor antigen binding domains from antibody libraries. An antibody havingthe identified antigen-binding domain (e.g., Fab) binding to the thirdantigen and the Fab domain of the dual Fab molecule can be obtained as amultispecific antibody by a multispecific antibody preparation methodgenerally known to those skilled in the art, for example, a method forpreparing an antibody having common L chains and two different H chains(technique controlling the interface of each domain in the Fc region),CrossMab, or Fab arm exchange. In other words, provided that a dual Fabmolecule can be identified, the desired multispecific antibody can beobtained according to a method generally known to those skilled in theart by combining Fab binding to the third antigen with the dual Fabbinding to the first and second antigens as shown in Example 9. TheseExamples showed that a molecule binding to CD3ε and the second antigencan be obtained by the adaptation of the dual Fab library to many typesof antigens, and further demonstrated that the molecule that can beobtained as described in Example 8 or 9 binds to the first antigen(CD3ε) and the second antigen, but does not bind to the first antigenand the second antigen at the same time. As mentioned above, Fab bindingto the third antigen can be identified by a method generally known tothose skilled in the art. Therefore, the desired antibody described inExample 1 can be obtained using the dual Fab library.

(9-5) CD3/Human CD154 Dual Fab Molecule

In Example 9, the obtained dual Fab molecule was shown to bind to CD3εand human CD154, but not bind to CD3ε and human CD154 at the same time.An antigen-binding domain binding to the third antigen can be furtheradded thereto by a method generally known to those skilled in the art.

In recent years, an agonistic antibody of a CD154 receptor CD40 has beenfound to enhance antitumor activity in a method of transferring cancerantigen-responsive T cells (J Immunother. 2012 April; 35 (3): 276-82).This Example revealed that the dual Fab molecule binding to CD3 andCD154 can be constructed. Anti-tumor effects mediated by CD40 can beexpected by selecting an antibody exhibiting agonist activity againstCD40 via CD154. In other words, this dual Fab can be expected to have Tcell-mediated cytotoxic activity by binding to CD3ε against cellsexpressing an arbitrary third antigen and to enhance antitumor effectsmediated by CD40 agonist signals via binding to CD154.

REFERENCE EXAMPLES [Reference Example 1] Preparation of AntibodyExpression Vector and Expression and Purification of Antibody

Amino acid substitution was carried out by a method generally known tothose skilled in the art using QuikChange Site-Directed Mutagenesis Kit(Stratagene Corp.), PCR, or In fusion Advantage PCR cloning kit (TakaraBio Inc.), etc., to construct expression vectors. The obtainedexpression vectors were sequenced by a method generally known to thoseskilled in the art. The prepared plasmids were transiently transferredto human embryonic kidney cancer cell-derived HEK293H line (InvitrogenCorp.) or FreeStyle 293 cells (Invitrogen Corp.) to express antibodies.Each antibody was purified from the obtained culture supernatant by amethod generally known to those skilled in the art using rProtein ASepharose™ Fast Flow (GE Healthcare Japan Corp.). As for theconcentration of the purified antibody, the absorbance was measured at280 nm using a spectrophotometer, and the antibody concentration wascalculated by use of an extinction coefficient calculated from theobtained value by PACE (Protein Science 1995; 4: 2411-2423).

[Reference Example 2] Evaluation of Binding of Antibody to ECM(Extracellular Matrix)

The binding of each antibody to ECM (extracellular matrix) was evaluatedby the following procedures with reference to WO2012093704 A1: ECMPhenol red free (BD Matrigel #356237) was diluted to 2 mg/mL with TBSand added dropwise at 5 μL/well to the center of each well of a platefor ECL assay (L15XB-3, MSD K.K., high bind) cooled on ice. Then, theplate was capped with a plate seal and left standing overnight at 4° C.The ECM-immobilized plate was brought to room temperature. An ECLblocking buffer (PBS supplemented with 0.5% BSA and 0.05% Tween 20) wasadded thereto at 150 μL/well, and the plate was left standing at roomtemperature for 2 hours or longer or overnight at 4° C. Next, eachantibody sample was diluted to 9 μg/mL with PBS-T (PBS supplemented with0.05% Tween 20). A secondary antibody was diluted to 2 μg/mL with ECLDB(PBS supplemented with 0.1% BSA and 0.01% Tween 20). 20 μL of theantibody solution and 30 μL of the secondary antibody solution wereadded to each well of a round-bottomed plate containing ECLDB dispensedat 10 μL/well and stirred at room temperature for 1 hour while shieldedfrom light. The ECL blocking buffer was removed by inverting the ECMplate containing the ECL blocking buffer. To this plate, a mixedsolution of the aforementioned antibody and secondary antibody was addedat 50 μL/well. Then, the plate was left standing at room temperature for1 hour while shielded from light. The sample was removed by invertingthe plate, and READ buffer (MSD K.K.) was then added thereto at 150μL/well, followed by the detection of the luminescence signal of thesulfo-tag using Sector Imager 2400 (MSD K.K.).

[Reference Example 3] Preparation of Human IgA

An Fc region in a naturally occurring human IgA sequence was used ashuman IgA (human IgA-Fc). For the purpose of biotinylating the Cterminus of the human IgA-Fc, a gene fragment encoding a specificsequence (AviTag sequence, SEQ ID NO: 79) for biotin ligase-mediatedbiotinylation was linked via a linker to a gene fragment encoding thehuman IgA-Fc. The gene fragment encoding a protein containing the humanIgA-Fc and the AviTag sequence linked (SEQ ID NO: 80) was integrated tovectors for expression in animal cells, and the constructed plasmidvectors were transferred to FreeStyle 293 cells (Invitrogen Corp.) using293Fectin (Invitrogen Corp.). In this operation, the cells werecotransfected with the expression vector and a gene encoding EBNA1 (SEQID NO: 81) and a gene encoding biotin ligase (BirA, SEQ ID NO: 82), andbiotin was further added for the purpose of biotinylating the humanIgA-Fc. The cells transfected according to the procedures mentionedabove were cultured at 37° C. for 6 days in an 8% CO₂ environment sothat the protein of interest was secreted into the culture supernatant.

The cell culture solution containing the human IgA-Fc of interest wasfiltered through a 0.22 μm bottle-top filter to obtain a culturesupernatant. The culture supernatant diluted with 20 mM Tris-HCl (pH7.4) was applied to HiTrap Q HP (GE Healthcare Japan Corp.) equilibratedwith 20 mM Tris-HCl (pH 7.4), followed by the elution of the humanIgA-Fc of interest with the concentration gradient of NaCl. Next, theHiTrap Q HP eluate diluted with 50 mM Tris-HCl (pH 8.0) was applied toSoftLink Avidin column (Promega Corp.) equilibrated with 50 mM Tris-HCl(pH 8.0), followed by the elution of the human IgA-Fc of interest with 5mM biotin, 150 mM NaCl, and 50 mM Tris-HCl (pH 8.0). Then, associateswere removed as undesired impurities by gel filtration chromatographyusing Superdex 200 (GE Healthcare Japan Corp.) to obtain purified humanIgA-Fc with the buffer replaced with 20 mM histidine-HCl and 150 mM NaCl(pH 6.0).

INDUSTRIAL APPLICABILITY

The present invention can enhance activity brought about byantigen-binding molecules and provides a polypeptide that can circumventthe cross-linking between different cells resulting from binding toantigens expressed on the different cells, which is considered to beresponsible for adverse reactions, and is suitable as a drug.

1-30. (canceled)
 31. A method for producing an antigen-binding moleculethat recognizes a first antigen and a second antigen different from thefirst antigen, the method comprising: identifying a startingantigen-binding molecule comprising a starting antibody variable regionthat recognizes the first antigen and does not recognize the secondantigen; preparing a library of altered antigen-binding moleculescomprising molecules that vary from the starting antigen-bindingmolecule and from each other by at least one amino acid alteration inthe antibody variable region; and selecting from the library an alteredantigen-binding molecule comprising an altered antibody variable regioncomprising a first antigen-binding site and a second antigen-bindingsite, wherein: (a) the first antigen-binding site recognizes the firstantigen, and (b) the second antigen-binding site recognizes the secondantigen, and (c) the first antigen-binding site cannot bind to the firstantigen when the second antigen-binding site is bound to the secondantigen, and (d) the second antigen-binding site cannot bind to thesecond antigen when the first antigen-binding site is bound to the firstantigen.
 32. The method according to claim 31, further comprising:culturing a cell comprising (i) nucleic acid encoding a polypeptide orpolypeptides comprising the altered antibody variable region and (ii)nucleic acid encoding a polypeptide or polypeptides comprising a secondantibody variable region that binds to a third antigen different fromthe first and second antigens, such that the cell expresses amultispecific antigen-binding molecule; and recovering the multispecificantigen-binding molecule from the cell.
 33. The method according toclaim 31, wherein the first antigen is naturally expressed on a firsttype of cell and the second antigen is naturally expressed on adifferent type of cell.
 34. The method according to claim 32, whereinthe multispecific antigen-binding molecule further comprises an antibodyFc region.
 35. The method according to claim 34, wherein the antibody Fcregion has reduced binding activity against at least one human FcγR,compared to binding to the at least one human FcγR by a naturallyoccurring human IgG1 antibody.
 36. The method according to claim 31,wherein the at least one amino acid alteration is at least onesubstituted or inserted amino acid.
 37. The method according to claim31, wherein at least one of the amino acid alterations is insertion of 1to 25 amino acids.
 38. The method according to claim 31, wherein atleast one of the amino acid alterations is at a position located withina CDR1, CDR2, CDR3, or FR3 region of the altered antibody variableregion.
 39. The method according to claim 31, wherein at least one ofthe amino acid alterations is at a position located within a loop of thealtered antibody variable region.
 40. The method according to claim 31,wherein at least one of the amino acid alterations is at a positionselected from heavy chain variable domain Kabat numbering positions 31to 35, 50 to 65, 71 to 74, and 95 to 102, and light chain variabledomain Kabat numbering positions 24 to 34, 50 to 56, and 89 to
 97. 41.The method according to claim 31, wherein either the first or the secondantigen is a molecule specifically expressed on the surface of a T cell,and the other of the first and second antigens is a molecule expressedon the surface of a T cell or any other immunocyte.
 42. The methodaccording to claim 31, wherein either the first or the second antigen isCD3.
 43. The method according to claim 42, wherein the other of thefirst and second antigens is selected from the group consisting of FcγR,TLR, lectin, IgA, an immune checkpoint molecule, a TNF superfamilymolecule, a TNFR superfamily molecule, and an NK receptor molecule. 44.A method for producing an antigen-binding molecule that recognizes afirst antigen that is human CD3 and a second antigen that is not humanCD3, the method comprising: identifying a starting antigen-bindingmolecule comprising a first antibody variable region that recognizes thefirst antigen and does not recognize the second antigen; preparing alibrary of altered antigen-binding molecules comprising molecules thatvary from the starting antigen-binding molecule and from each other byat least one amino acid alteration in the first antibody variableregion; and selecting from the library an altered antigen-bindingmolecule comprising an altered first antibody variable region comprisinga first antigen-binding site and a second antigen-binding site, wherein:(a) the first antigen-binding site recognizes the first antigen, and (b)the second antigen-binding site recognizes the second antigen and doesnot recognize human CD3, and (c) the first antigen-binding site cannotbind to the first antigen when the second antigen-binding site is boundto the second antigen, and (d) the second antigen-binding site cannotbind to the second antigen when the first antigen-binding site is boundto the first antigen.
 45. The method according to claim 44, furthercomprising: culturing a cell comprising (i) nucleic acid encoding apolypeptide or polypeptides comprising the altered antibody variableregion and (ii) nucleic acid encoding a polypeptide or polypeptidescomprising a second antibody variable region that binds to a thirdantigen different from the first and second antigens, such that the cellexpresses a multispecific antigen-binding molecule comprising thealtered antibody variable region and the second antibody variableregion; and recovering the multispecific antigen-binding molecule fromthe cell.
 46. The method according to claim 44, wherein the secondantigen is naturally expressed on a cell that is not a T cell.
 47. Themethod according to claim 44, wherein the second antigen-binding site islocated within a loop or a CDR1, CDR2, CDR3, or FR3 region of the firstantigen-binding site.
 48. The method according to claim 44, wherein thesecond antigen-binding site is located within a CDR3 of the firstantigen-binding site.
 49. The method according to claim 45, wherein themultispecific antigen-binding molecule further comprises an antibody Fcregion.
 50. The method according to claim 49, wherein the antibody Fcregion exhibits reduced binding to at least one human FcγR, compared tobinding to the at least one human FcγR by a naturally occurring humanIgG1 antibody.
 51. The method according to claim 44, wherein the secondantigen is selected from the group consisting of FcγR, TLR, lectin, IgA,an immune checkpoint molecule, a TNF superfamily molecule, a TNFRsuperfamily molecule, and an NK receptor molecule.
 52. The methodaccording to claim 44, wherein the second antigen-binding site is apeptide.
 53. The method according to claim 52, wherein the peptide bindsto an antigen selected from the group consisting of vascular endothelialgrowth factor receptor (VEGFR), tumor necrosis factor receptor (TNFR),toll-like receptor (TLR) 5, TLR4, TLR2, T cell very late antigen (VLA)receptor, platelet-derived growth factor receptor (PDGFR), Naip5Nod-like receptor (NLR), integrin, FcγRIIa, epidermal growth factorreceptor (EGFR), death receptor (DR) 5 agonist, C—X—C chemokine receptortype 4 (CXCR4), cluster of differentiation (CD) 40, and CD154.
 54. Themethod according to claim 44, wherein the second antigen-binding site islocated in the first antibody variable region of the alteredantigen-binding molecule, at a position selected from heavy chainvariable domain Kabat numbering positions 31 to 35, 50 to 65, 71 to 74,and 95 to 102, and light chain variable domain Kabat numbering positions24 to 34, 50 to 56, and 89 to
 97. 55. The method according to claim 45,wherein the third antigen is a molecule specifically expressed in acancer tissue.
 56. The method according to claim 45, wherein themultispecific antigen-binding molecule is a sc(Fv)2.
 57. The methodaccording to claim 45, further comprising conjugating the multispecificantigen-binding molecule with polyethylene glycol (PEG) or an anticanceragent.
 58. The method of claim 44, wherein the altered first antibodyvariable region comprises a heavy chain variable domain amino acidsequence comprising, at each of the following positions (all by Kabatnumbering), one of the amino acid residues indicated for that position:31: Ile, Asn, or Ser; 32: Ala; 33: Trp; 34: Met; 35: His; 50: Gln; 51:Ile; 52: Lys; 52(a): Asp; 52(b): Arg or Lys; 52(c): Ala, Gly, Leu, Gln,Val, or Ser; 53: Gln, Ser, or Asn; 54: Ala, Gly, Leu, Gln, Ser, or Asn;55: Tyr; 56: Leu, Asn, or Ala; 57: Ala, Asn, or Thr; 58: Tyr; 59: Tyr;60: Ala; 61: Pro or Glu; 62: Ser; 63: Val; 64: Lys; 65: Gly; 95: Val;96: His; 97: Tyr; 98: Ala, Gly, Pro, Ser, Thr, Gln, or His; 99: Ala,Leu, Ser, Gln, or Thr; 100: Ala, Val, Gly, Ser, Thr, Tyr, or Phe;100(a): Ala, Val, Gly, Ser, Thr, Tyr, Phe, or Asp; 100(b): Gly, Ser,Thr, Tyr, Phe, or Asp; 100(c): Ala, Val, Leu, Gly, Ser, Tyr, or Phe;100(d): Leu, Gly, Ser, Tyr, or Phe; 100(e): Pro, Gly, Ser, Tyr, or Phe;100(f): Ala, Ser, Gln, Lys, Tyr, or Gly; 100(g): Tyr, Phe, or Gly;100(h): Gly; 100(i): Val; 101: Asp; 102: Ile.
 59. The method of claim44, wherein the altered first antibody variable region comprises a lightchain variable domain amino acid sequence comprising, at each of thefollowing positions (all by Kabat numbering), one of the amino acidresidues indicated for that position: 24: Arg, Ala, Glu, Gly, Gln, Ser,Thr, or Tyr; 25: Ser Ala, Pro, or Thr; 26: Ser or Asp; 27: Gln, Asp, orGlu; 27(a): Ser, Glu, or Pro; 27(b): Leu, Ile, Pro, or Val; 27(c): Valor Leu; 27(d): His; 27(e): Ser, Ala, Gly, Ile, Leu, Met, Asn, Pro, Gln,Thr, or Val; 28: Asn; 29: Arg; 30: Asn, Phe, His, Met, Gln, or Tyr; 31:Thr or Ile; 32: Tyr; 33: Leu, Ala, Ile, or Val; 34: His, Ala, Gly, Thr,or Val; 45: Gln; 50: Lys; 51: Val, Gly, or Ile; 52: Ser, Ala, Gln, orTyr; 53: Asn, Pro, or Trp; 54: Arg or Pro; 55: Phe, Ala, Gly, His, Lys,Asn, Pro, or Tyr; 56: Ser, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Thr,Val, or Tyr; 74: Lys or Thr; 77: Arg or Ser; 79: Gly, Ala, Asn, or Thr;90: Gln or Glu; 91: Gly; 92: Thr or Ser; 93: Gln, Ala, or Ser; 94: Val,Ala, Asp, Asn, Ser, or Thr; 95: Pro; 96: Tyr or Phe; 97: Thr; 107: Lysor Glu.
 60. The method according to claim 44, wherein the secondantigen-binding site comprises an amino acid residue or a peptide thatis inserted in the first antibody variable region between a pair ofpositions selected from the following pairs (all by Kabat numbering):heavy chain variable domain positions 52b and 52c, heavy chain variabledomain positions 72 and 73, heavy chain variable domain positions 98 and99, heavy chain variable domain positions 100 and 100a.
 61. A method ofproducing a multispecific antigen-binding molecule that recognizes afirst antigen that is human CD3, a second antigen that is not human CD3,and a third antigen different from the first antigen and second antigen,the method comprising: providing nucleic acid encoding (i) a polypeptideor polypeptides comprising a heavy chain variable domain and a lightchain variable domain that together comprise a first antibody variableregion comprising a first antigen-binding site and a secondantigen-binding site, and (ii) a polypeptide or polypeptides comprisinga heavy chain variable domain and a light chain variable domain thattogether comprise a second antibody variable region comprising a thirdantigen-binding site, wherein: (a) the first antigen-binding siterecognizes the first antigen, (b) the second antigen-binding siterecognizes the second antigen and does not recognize human CD3, (c) thefirst antigen-binding site cannot bind to the first antigen when thesecond antigen-binding site is bound to the second antigen, (d) thesecond antigen-binding site cannot bind to the second antigen when thefirst antigen-binding site is bound to the first antigen, and (e) thethird antigen-binding site recognizes the third antigen; expressing thenucleic acid, to produce the multispecific antigen-binding molecule; andrecovering the multispecific antigen-binding molecule.